ELN - User contributions [en]

Web passwords

2023-06-29T09:42:36Z

Paul.xu:

FDA: A8j*3?

Chineseantibody.org: p*c*b*

[https://portal.census.gov/surveys https://portal.census.gov/surveys]

paulshue@gmail.com/1Qaz@wsx8888

'''godaddy:'''

19729324/P*c*b*8*** excellgen.com

215901242  crm@excellgen.com 215901242 /P*c*b*8*** pureproteinlab.com

[https://dcc.godaddy.com/manage/pureproteinlab.com/dns https://dcc.godaddy.com/manage/pureproteinlab.com/dns]

SSL: excellgen, P*c*b*1! [https://secure.instantssl.com/ https://secure.instantssl.com]

ssl for pureproteinlab.com [https://dash.cloudflare.com,&nbsp https://dash.cloudflare.com,&nbsp]; crm@excellgen.com

[https://www.digicert.com/secure/ &nbsp https://www.digicert.com/secure/ &nbsp];   lab@excellgen.com Username excellgen  pw: P*c*b*1!

godaddy excellgenbio.com, phs.phd@gmail.com/p*c*b*8888, 251919351

SSSL

[https://www.ssls.com/user/orders https://www.ssls.com/user/orders] paulshue@gmail.com

'''FedEx'''

295408952

excellgensales/A*j*1?

'''Wireless'''

vprotein

'''storage 192.168.1.4 MYBOOKLIVE'''

vprotein

'''Comcast'''

Admin page: [http://10.1.10.1 http://10.1.10.1] User name: cusadmin Password: highspeed

 '''Phone 1:'''

[http://10.1.10.10/ http://10.1.10.10/] paulshue(excellgen)

'''craigslist'''

phs.phd@gmail.com/1qaz-plm

'''Phone.com'''

innovita/1qaz2wsx
[[Category:Contacts]] [[Category:Paul]] [[Category:Company]]

Vendor accounts

2023-06-29T09:33:33Z

Paul.xu:

== FedEx 295408952 ==

innovita/A*j*10*

excellgensales/A*j*5?

account # 295408952, last 4 digit: 2005

meter id: 0945471

old: 307640961

control #: 0621023651

billing 16224 Orchard View Ct, Gaithersburg, MD 20878, us 1.800.463.3339 Fedex shipping manager: 0945471
<div>'''Development'''</div><div>Required for All Web Services</div><div>Developer Test Key: PX6njeHw9rEKZMKR</div><div>Required for FedEx Web Services for Intra Country Shipping in US and Global</div><div>Test Account Number: 510087380 </div><div>TestMeter Number: 118895091 </div><div>Required for FedEx Web Services for Office and Print</div><div>TestFedEx Office Integrator ID: 123</div><div>TestClient Product ID: TEST</div><div>TestClient Product Version: 9999</div><div> </div><div>'''Production'''</div><div>Account number:295408952 </div><div><div>Supported Web Services: FedEx Web Services for Shipping</div><div>Authentication Key: fuClHpTw0TUDkzs8</div><div>Meter Number: 111636762</div><div>(old: 0945471) </div></div>

== UPS ==

excellgen/1qaz-plm UPS Account Number: 51802E

DHL

shipping@excellgen.com/!Qaz2wsx 

Go-dave: excellgensales/P*c*b*8888, lab@excellgen.com

== ATCC ==

Account Number: 199255

(800) 638-6597

paul.xu@excellgen.com/Alexjessica1!

[http://www.atcc.org/ http://www.atcc.org/]

== Agilent.com ==

innovita 1@#$qWER

== Bio-Rad ==

paul.xu@excellgen.com/1qaz2wsx, 1026414

== '''Invitrogen''' ==

maryjo.laughlin@lifetech.com tax exempt

account #: 68976453

gene synthesis: paulshue@gmail.com/1qaz2wsx

== emdmillipore  ==

crm@excellgen.com/1qaz2wsx

== '''Lampire BIological products''' ==

[http://www.lampire.com/cart_thanks.php http://www.lampire.com/cart_thanks.php]

username: paul.xu@excellgen.com

password: 123456

== BioTangusa.com ==

excellgen/1234qwer

== IDT Oligo ==

Login Name: excellgen Password: 1qaz2wsx [https://www.idtdna.com/orderstatus/default.aspx https://www.idtdna.com/orderstatus/default.aspx]

== '''Clontech''' ==

Account #: 32963

800-662-2566, opt 1

email: orders@clontech.com

fax: 800-424-1350

== Fermentas ==

[http://www.fermentas.com http://www.fermentas.com], [http://www.fermentas.com/fd/go.php http://www.fermentas.com/fd/go.php]

tel: +1 800 3409026

Christina.Pavlick@thermofisher.com

== EMD Millipore ==

EMD Millipore 290 Concord Road 01821 Billerica, MA Phone: 781 533-6000 Fax: +49 6151 72 2000 E-mail: Customer.CareDesk@emdchemicals.com

paulshue@gmail.com/1234qwer

== '''NEB''' ==

paul.xu@excellgen.com/1qaz@WSX

== ATCC ==

paul.xu@excellgen.com/1qaz@WSX

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== '''Beckman''' ==

Account #: 724179

billto/ship to : 1908928

 excellgen /A*j81!

Net 30

Remit To: Beckman Coulter, Inc.. Dept. CH 10164 Palatine, IL 60055-0164

cat#: 355622 for tube

Customer Service line 1-800-526-3821 or fax 800-232-3828

tech support: 800-369-0383

Invoice Copies request. Dial 800-526-3821 select A/R opt 5 select invoice copies opt 1--leave your request with fax number to reply to.

 CREDIT & COLLECTION U.S. Phone (800) 526-3821 opt 5 Dept fax (714) 459-1000 creditcollectionsus@Beckman.com BECKMAN COULTER INC. 250 S. KRAEMER BLVD M/S M28 BREA, CA, 92822-0550

Centrifuge  Bottle & Cap Assembly #355622, Bottle, Assembly, Polycarbonate, 70 mL, 38 x 102 mm, 1-1/2 x 4 in, Aluminum Cap (qty. 6) $386

alum cap #339165, $31

Polycarbonate plug #334545 $9.00

pump oil #341661, 1L. $28.50

o-ring: 870384

O-ring for Type 35 rotor: small 11757, large 801767, new part numbers:  small

Ultracentrifuge tubes 344058

Ultracentrifuge tubes: 355655 38 × 102

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Bottle 
| Dimensions (mm) 
|  Cap Assembly 
| Bottle and Cap 
| Volume (mL) 
| Roter
| Maximum Speed 
|-
|
[https://www.beckman.com/supplies/tubes-and-bottles/bottles/355655 355655]

[https://www.beckman.com/supplies/tubes-and-bottles/bottles/355655 Polycarbonate Bottle], $55/6tubes

| 38 × 102 
| 355623 aluminum 
| 355622 
| 70 
| Type 35 
| 35 000 
|-
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
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|}

== Biologix ==

[http://www.biologixresearch.com http://www.biologixresearch.com], excellgen/1qaz2wsx

== Kapa ==

Kapa HiFi, KK2502,  $225.00, Phone: 781-497-2933, quote KAPA-9250

SurveyMonkey

excellgen/1234qwer

== Eurofinsgenomics ==

excellgenoligos/1Qaz2wsx

== '''Pierce''' ==

[http://www.piercenet.com http://www.piercenet.com]

paul.xu@excellgen.com/1234qwer

== '''GE Pharmacia''' ==

[http://www.gelifesciences.com http://www.gelifesciences.com] [http://www.gelifesciences.com/aptrix/upp01077.nsf/content/life-sciences_homepage http://www.gelifesciences.com/aptrix/upp01077.nsf/content/life-sciences_homepage] Tel: 1-800-526-3593 (select option 2)

paul.xu@excellgen.com p*c*b*8*

== '''Sigma''' ==

Excellgen/p*c*b*8888

== spectrumlabs ==

excellgen/1qaz2wsx

== '''ABI''' ==

[https://products.appliedbiosystems.com https://products.appliedbiosystems.com]

excellgen/1qaz2wsx

== '''VWR''' ==

paul.xu@excellgen.com/protein
crm@excellgen.com/excellgenvwr8888
[https://www.vwr.com https://www.vwr.com] 3174081

shipping@excellgen.com/p*c*b*8888,  80596482

== '''KPL''' ==

paul.xu@excellgen.com/12345678, Account 363737 [https://www.kpl.com https://www.kpl.com]

== '''Qiagen''' ==

 account # 307460 800-426-8157

== '''Cellgro''' ==

account # 3369 800-235-5476 E-mail: paul.xu@excellgen.com Password: 1234qwer quote #: 00000331, 10-013-CV; 6 x 500 mL; DMEM 3.00 $36.00 DMEM 10-013-CV, -CM, -LB, -LX contain 4.5 g/L glucose, L-glutamine and sodium pyruvate. Quote Number 00000764, 30-001-CI; 6 x 100 mL; PEN-STREP 1.00 $45.00, 21-030-CV; 6 x 500 mL; DPBS 1.00 $35.00 Quote Number 00000331: 25-054-CI; 6 x 100 mL; TRYPSIN 1.00 $60.00

== '''Systembio''' ==

[https://www.systembio.com https://www.systembio.com]

paulshue/1234qwer

Direct: 650-968-2200 Toll Free: 888-266-5066 Fax: 650-968-2277

== '''Invivogen''' ==

paulshue@gmail.com, 1234qwer

== '''Lucigen''' ==

[https://www.lucigen.com https://www.lucigen.com]

paulshue/1234qwer

== '''amresco-inc.com''' ==

paul.xu@excellgen.com/1234qwer

Uline: 7003572

== '''Polysceinces''' ==

[https://www.polysciences.com https://www.polysciences.com], paulshue@gmail.com/1234qwer

== '''RedCross''' ==

00131241/1QAZ2wsx

[https://ic2.redcross.org/ https://ic2.redcross.org/]

== p212121 ==

L Arabinose,  $199/1 KG, Puromycin 100mg/$95, Ampicillin 149/100g, Carbenicillin, 136/25g

[http://store.p212121.com http://store.p212121.com]

== '''Macrogen''' ==

[http://www.macrogenusa.com/ http://www.macrogenusa.com/]

excellgen/1234qwer

== '''grants.gov and CCR.gov''' ==

Grants: excellgen/1qaz)OKN

DUNS+4: 805936957000

8059369570000/1qaz)OKN

CCR: excellgen/1qaz)OKN

MPIN: pinxu2085

== W2 form ==

[https://secure.ssa.gov/acu/IRESWeb/iresRegistration.do https://secure.ssa.gov/acu/IRESWeb/iresRegistration.do]

userid: YHD53YCD

password: 1234qwer

excellgen 1Qaz@wsx

www.chemimpex.com paul.xu@excellgen.com/1qaz2wsx"

== Alfa Aesar ==

[https://www.alfa.com https://www.alfa.com], paul.xu@excellgen.com 1qaz2wsx

== ThermoFisher ==

[http://www.labequipmentparts.com http://www.labequipmentparts.com], paul.xu@excellgen.com

[https://www.beiresources.org/Home.aspx https://www.beiresources.org]  paul.xu@excellgen.com A*j*1!

== Excellgen ==

== '''Excellgen, Inc.''' ==

'''15601 Crabbs Branch Way Lab W204 Rockville, MD 20855-2743 Phone: 240-454-9768 Fax: 240-454-9778'''

Tax ID: 26-1935374

DUNS: 805936957

Credit Card: Paul Xu, MasterCard, 5474-1516-9749-3416, exp 01/13, 5839.

Credit card address (if ask): 16224 Orchard View Ct, Gaithersburg, MD, 20878, 202-997-6778

Bank Bank of America Account Name Excellgen Account Number 4460 0855 4109 Routing Number 026009593 SWIFT Code BOFAUS3N Address 1048 Quince Orchard Rd, Gaithersburg   Maryland, 20878, USA Phone  1-301-963-5350
[[Category:Suppliers]] [[Category:Company]]

Passwords

2023-05-17T14:31:07Z

Paul.xu: Replaced content with "<parsererror style="display: block; white-space: pre; border: 2px solid #c77; padding: 0 1em 0 1em; margin: 1em; background-color: #fdd; color: black"> === This page conta..."

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Sodium Borate

2022-10-31T22:08:39Z

Paul.xu:

Preparation of 20X Fast sodium borate buffer for gel electrophoresis

Sodium borate buffer (200mM NaOH, pH ~8.2) 1. Add 8g NaOH (MW=40), 47g boric acid (MW=61.83) to 900ml distilled water, make sure all powers dissolve completely. 2. Make final volume to 1L by adding water.  pH should be around 8.2.
[[Category:Buffer Recipe]]

T7 High Yield In vitro Translation

2022-10-08T16:45:35Z

Paul.xu:

== Materials ==

• S30 T7 High Yield In vitro Protein Expression from Promega

• DNase- and RNase-free 1.5ml microcentrifuge tubes

• plasmid DNA encoding the protein of interest

• floor incubator shaker or thermomixer

1. Remove the reagents from the freezer, and allow them to thaw on ice or in an ice-water bath.

2. Set up the following reactions on ice in a DNase- and RNase-free 1.5ml microcentrifuge tube:

Mix thoroughly by pipetting several times or vortexing gently, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube.

3. Mix thoroughly by pipetting several times or vortexing gently, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube

4. Quickly bring reaction to 37°C, and incubate the reactions with vigorous shaking for 1 hour (see Notes 12 and 13).

5. Stop the reaction by placing the tubes in an ice-water bath for 5 minutes.

6. Analyze the results of the reaction. See Section 6 for gel analysis of proteins. To evaluate the results of the positive control reaction, see Section 7.

Notes:

1. The reaction is very sensitive to nuclease contamination. Wear gloves, and use RNase- and DNase-free reagents and reaction tubes, and filtered pipette tips.

2. Higher protein expression levels can be achieved by increasing the amount of T7 S30 Extract, Circular, in the reaction (up to 45% of the reaction volume) while keeping the total volume constant. However, increasing the volume of the extract will reduce the total number of reactions.

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Component 
| Standard Reaction 
|
Negative Control

(see Note 10)

|
Positive Control

(see Note 11)

|-
| DNA template (see Notes 7–9) 
| 1µg 2016, 2060 
| 0 
| 2µl 
|-
| S30 Premix Plus (mix well prior to use)  
| 20µl 
| 20µl 
| 20µl 
|-
| T7 S30 Extract, Circular (mix gently prior to use) 
| 18µl 
| 18µl 
| 18µl 
|-
| Nuclease-Free Water to a final volume of 
| 50µl 
| 50µl 
| 50µl 
|}

2060 pET30b-Tat-Flp-ecoli-6H , E coli Flp recombinase, His tag is at C terminus, no His tag at N terminus

MGRKKRRQRRRPPAGTSVSLKKKRKVSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRILEHHHHHH

2106 pET28b-iFLP

MGHHHHHHGMGAAGRKKRRQRRRPPAGTSVSLKKKRKVSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI

3. Template DNA and water purity are critical. If protein synthesis efficiencies are low, examine the quality of the template DNA and water. Read the discussion concerning DNA templates in Section 3.A.

4. If necessary, transcription by the endogenous E. coli RNA polymerase can be inhibited by the antibiotic rifampicin, while transcription by the phage T7 RNA polymerase is unaffected. To inhibit the endogenous RNA polymerase, add 1µl of 50µg/ml of rifampicin in water (Section 10.A) prior to adding the DNA template to the reaction. Adding excess rifampicin is unnecessary and may decrease protein synthesis levels.

5. To obtain fluorescently labeled or biotinylated protein, use 2µl of FluoroTect™ or Transcend™ tRNA in 50µl of reaction. Titrate the tRNAs if necessary. For incorporation of radioactive amino acids, we recommend using the E. coli T7 S30 Extract System for Circular DNA (Cat.# L1130).

6. The reaction volume can be scaled down to as little as 5µl without reducing protein yields. For reactions less than 25µl, we recommend assembling the reactions in PCR strip tubes or 96-well PCR plates; for reactions of 25–50µl, we recommend using 0.5ml microcentrifuge tubes; for reactions of 50–250µl, we recommend using 1.5ml microcentrifuge tubes.

7. The amount of DNA required might be template-dependent. We recommend 0.5–1µg/50µl reaction of <5kb plasmid DNA with a T7 promoter. Higher DNA concentrations (such as 2µg/50µl reaction) can be used for large plasmids and vectors with an E. coli promoter. If necessary, optimize the amount of DNA added. In general, reactions should not contain more than 4µg of DNA. An increased amount of DNA can increase the incidence of internal translational initiation or the number of prematurely arrested translation products. 

8. Refer to Section 3.A for a discussion on suitable DNA templates for this system.

9. The T7 S30 Extract, Circular, contains nuclease activity, preventing the use of linear DNA templates, such as PCR products. PCR products containing a T7 promoter and a ribosome binding site can be expressed using TnT® T7 Quick for PCR DNA (Cat.# L5540).

10. For a negative control, omit the DNA from the reaction. Use the negative control to determine the protein expression background in downstream analysis.

11. Use the S30 T7 Control DNA to synthesize Renilla luciferase, which has been codon optimized for use with mammalian cells. The expressed hRluc protein (37kDa) can be detected by Coomassie® blue staining of polyacrylamide gels following electrophoresis (Figure 2, Panel A, lane 2). The calculated molecular weight (MW) of hRluc protein is 37kDa, but it migrates with an apparent MW of ~33kDa.

12. The reaction may be incubated at 24–37°C. The fastest rate occurs at 37°C for approximately 1 hour, although the reaction will continue for several hours at a slower rate. Lower temperatures produce a slower rate of translation but often extend the time to several hours. Enhanced expression at lower temperatures for longer times appears to be gene- or protein-specific and may be tried if the standard reaction at 37°C for 1 hour does not produce the desired results.

13. To achieve maximum protein expression, quickly bring the reactions to the target temperature (e.g., place the reactions in a heat block preset to target reaction temperature), and shake the reactions vigorously at 300rpm with a floor incubator shaker or in a thermomixer at 1,200 rpm. 

== Purification of His- or HQ-Tagged Recombinant Protein Using Magnetic Ni-Particles ==

Materials

• magnetic stand (see Section 10.C for options)

• Magnetic Ni Beads

• solid NaCl or 5M NaCl solution

1. Add 50µl of Binding/Wash Buffer to a 50µl reaction containing the His-tagged protein. [For HQ-tagged protein, also add NaCl (500mM final concentration) to improve binding of HQ tag to magnetic Ni-Particles.]

2. Vortex the magnetic Ni-Particles to obtain a uniform suspension.

3. Add 30µl of magnetic Ni-Particles.

Note: You may need to titrate the amount of magnetic Ni-Particles used for proteins expressed at higher or lower levels.

4. Invert tube to mix (approximately 10 times), and incubate for 2 minutes at room temperature. Make sure the magnetic Ni-Particles are well mixed.

5. Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the magnetic Ni-Particles. Using a pipette, carefully remove the supernatant.

6. Remove the tube from the magnetic stand. Add 150µl of magnetic Binding/Wash Buffer with 500mM NaCl to the magnetic Ni-Particles, and pipet to mix. Make sure that particles are resuspended well.

7. Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the magnetic Ni-Particles. Using a pipette, carefully remove the supernatant.

8. Repeat the wash step twice for a total of three washes.

9. Remove the tube from the magnetic stand. Add 100µl of magnetic Elution Buffer, and pipet to mix. Note: HQ-tagged proteins may elute at a lower concentration of imidazole (50–100mM) compared to polyhistidine-tagged proteins. The magnetic Elution Buffer, which contains 500mM imidazole, can be diluted with magnetic Binding/Wash Buffer or water to decrease the imidazole concentration, if desired.

10. Incubate for 1–2 minutes at room temperature. Place tube in a magnetic stand to capture the magnetic Ni-Particles. Using a pipette, remove the supernatant containing the purified protein.

Analyze the samples by SDS-PAGE or by functional assay. Note: Steps 8–10 can be omitted, and the sample containing the magnetic Ni-Particles can be used directly for SDS-PAGE analysis. Add 30µl of 1X SDS gel-loading buffer to resuspend the magnetic Ni-Particles. Load the sample directly onto an SDS-polyacrylamide gel.
[[Category:Protocols]]

6X Loading Dye

2022-10-04T14:25:03Z

Paul.xu:

== 1% Cresol Red Dye Stock yield 50 ml ==

Add 500 mg to 50 ml of ddH2O in a 50 ml tube or bottle

Shake to dissolve,Store at room temperature

== Cresol Red Loading Dye containing 0.02% Cresol Red ==

yield 100 ml of working solution Add 30 g of glycerol to ~70 ml of ddH2O Shake to dissolve Add 2 ml of 1% Cresol Red Dye or amaranth, adjust to 100 ml Shake tube to mix. Store at 4°C

== Protocol 2 ==

*300 ml glycerol (30%)
*2.5 g bromophenol blue or '''Cresol Red '''(0.25%)
*dH2O to 1000mL

== 10X Xylene Cyanol/Bromophenol Blue DNA loading buffer recipe ==

*0.025 g of Xylene Cyanol FF
*0.025 g of Bromophenol Blue0.025 g cresol red (optional)
*1.25 ml of 10% SDS (optional)
*4 ml 0.5 M EDTA (optional)
*2 ml 1 M Tris-Cl pH 7.6-8.0 (optional)
*12.5 ml of glycerol
*water to 20 ml Store sucrose buffers at 4 or -20C.
[[Category:Buffer Recipe]]

Sequence Protocol

2022-07-15T15:04:03Z

Paul.xu:

Psomagen Sanger sequencing protocol:

Plasmid and primer separately: 150 ng/ul plasmid x 10 ul, 10  uM primer x 5 ul;

Premix: 100 ng/ul x 5 ul plamid containing 5 pmol primer (total amount).

2956

2022-05-15T00:49:37Z

Paul.xu: Paul.xu moved page 2956 to 2966

#REDIRECT [[2966]]

2966

2022-05-15T00:49:36Z

Paul.xu: Paul.xu moved page 2956 to 2966

WO2010099296 Hyperactive PiggyBac transposases

catatgaagctTAATACGACTCACTATAagGATTAAT

pfizer 5'UTR

gagaataaactagtattcttctggtccccacagactcagagagaaccc

Kozak

gccacc

ATGGGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGCGAGGACAGCGACAGCGAGATCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACAAGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGGCCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCGGCGCCACCTTCAGAGACACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCACCGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCAGATGCCCCTTCAGAATGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGGCACCAAGTACATGATCAAC

change GGCATGC to GGaATGC to remove Sph1

GGaATGC

CCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACAACTGGTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGCGGGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGGACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAACATCAGCAACATCCTGCCCAACGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTC
<pre>Stop</pre>

TGA
<pre>TGA
3’ UTR
Xho1 Not1
CTCGAG CGGCCGC
change C to g to remove Sph1 site
CTGGTACTGCATGgACGCAATGCTAGCT GCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGC TAGTTCCAGACACCTCC

CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTT AGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCAC

ACCCTGGAGCTAGCA
</pre>
<pre />
Sph1

GCATGC

====

sequence for gene synthesis

CATATGAAGCTTAATACGACTCACTATAAGGATTAATGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGCGAGGACAGCGACAGCGAGATCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACAAGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGGCCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCGGCGCCACCTTCAGAGACACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCACCGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCAGATGCCCCTTCAGAATGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGGCACCAAGTACATGATCAACGGAATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACAACTGGTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGCGGGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGGACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAACATCAGCAACATCCTGCCCAACGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGATGACTCGAGCGGCCGCCTGGTACTGCATGGACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAGCATGC

MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

genewiz optimized

ATGGGCTCTTCACTGGATGACGAGCACATCTTGTCGGCACTGCTGCAGAGCGATGACGAA CTGGTGGGAGAGGACAGCGACAGTGAGATATCTGACCACGTGTCTGAGGATGATGTGCAG TCTGACACCGAGGAGGCGTTCATTGACGAAGTCCATGAGGTGCAACCGACCTCCTCCGGT TCGGAGATCCTGGACGAACAGAACGTGATTGAGCAACCAGGCTCCTCTCTCGCTAGCAAT CGAATCCTCACCCTGCCGCAGCGGACCATCCGCGGAAAAAACAAGCACTGTTGGTCAACG TCCAAGTCTACGCGGCGCTCGAGGGTCTCGGCACTAAACATAGTACGATCCCAGCGCGGG CCCACTAGGATGTGCCGCAACATCTACGATCCGTTGCTGTGCTTCAAACTATTTTTCACC GACGAGATCATCTCTGAGATCGTGAAATGGACGAACGCCGAGATTTCTCTGAAGCGCCGC GAGAGTATGACCGGCGCCACGTTCAGGGATACCAACGAGGACGAGATCTACGCCTTCTTC GGAATCCTGGTCATGACAGCCGTGCGCAAGGACAACCATATGTCGACGGATGACCTTTTT GACCGCTCTCTGTCCATGGTGTACGTGTCTGTGATGTCGCGGGACCGCTTCGACTTCCTG ATCCGCTGCCTGCGCATGGATGACAAGTCGATCCGCCCCACACTCAGAGAGAACGACGTG TTCACCCCGGTGCGAAAGATCTGGGACTTGTTCATCCACCAGTGTATCCAGAACTACACC CCTGGCGCGCACCTGACCATTGATGAGCAGCTGCTCGGCTTCCGTGGCCGTTGTCCCTTC CGCATGTATATTCCCAACAAGCCATCGAAGTACGGCATTAAGATCCTGATGATGTGCGAC AGCGGTACCAAGTACATGATCAACGGTATGCCCTATCTGGGGCGGGGCACCCAGACCAAC GGCGTTCCGCTTGGGGAGTACTACGTGAAGGAGCTGTCCAAGCCCGTGCATGGTTCCTGC CGAAACATCACCTGTGACAACTGGTTTACCTCTATCCCTCTGGCGAAGAACCTGCTCCAG GAGCCATACAAACTGACCATCGTTGGTACCGTGAGGTCCAATAAGCGCGAGATCCCGGAG GTTCTGAAGAATTCCCGCTCCCGCCCCGTGGGGACTTCCATGTTCTGCTTTGACGGCCCT TTGACCCTTGTGAGCTACAAGCCCAAACCTGCCAAGATGGTCTACTTACTGAGTAGTTGC GACGAAGATGCTAGCATCAATGAGAGCACTGGCAAGCCTCAGATGGTGATGTATTACAAC CAGACTAAAGGCGGCGTCGACACTCTAGACCAGATGTGCTCTGTAATGACGTGCTCCCGC AAAACAAATCGTTGGCCCATGGCTCTCCTGTATGGAATGATCAATATCGCCTGTATCAAC TCCTTCATCATCTACTCCCACAACGTGTCCTCTAAGGGCGAGAAGGTCCAGTCCCGGAAG AAATTTATGCGTAATCTTTACATGTCACTTACCTCCAGCTTTATGAGAAAGCGCCTGGAG GCTCCTACCCTTAAGCGTTACTTGCGCGACAACATCAGCAACATCCTGCCTAACGAGGTA CCCGGGACTAGCGATGACTCTACTGAGGAGCCCGTCATGAAAAAACGTACTTACTGTACC TACTGCCCATCCAAGATTCGTCGTAAGGCCAACGCGTCCTGCAAGAAGTGCAAGAAGGTC ATCTGCCGCGAGCACAACATTGACATGTGCCAGAGCTGCTTC

MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF
[[Category:Cloning]]

2957

2022-05-15T00:49:14Z

Paul.xu: Paul.xu moved page 2957 to 2967

#REDIRECT [[2967]]

2967

2022-05-15T00:49:14Z

Paul.xu: Paul.xu moved page 2957 to 2967

T7AG  Hyperactive PiggyBac transposases

subclone 2956 into 2953 using Nde1-Sph1

TAATACGACTCACTATAAGGtcttctggtccccacagactcagagagaacc

   CATATGAAGCTTAATACGACTCACTATAAGGATTAATGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGCGAGGACAGCGACAGCGAGATCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACAAGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGGCCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCGGCGCCACCTTCAGAGACACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCACCGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCAGATGCCCCTTCAGAATGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGGCACCAAGTACATGATCAACGGAATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACAACTGGTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGCGGGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGGACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAACATCAGCAACATCCTGCCCAACGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGATGACTCGAGCGGCCGCCTGGTACTGCATGGACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAGCATGC

 CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC  

[[Category:Cloning]]

MRNA Products

2022-05-05T00:06:31Z

Paul.xu:

{| border="1" cellspacing="1" cellpadding="1" style="width: 881px;"
|-
| Product Name 
| Plasmid Name
| style="width: 66px;" | Cat #
| style="width: 13px;" | Clone #
| Plasmid E coli stock
| style="width: 40px;" | mRNA Length
| style="width: 375px;" | Digestion or PCR
| style="width: 259px;" | Note
|-
| Cre
| T7-VEE-T7AG_Cre 
| style="width: 66px;" | 
| style="width: 13px;" | 2893 
| 35/F4 
| style="width: 40px;" | 1610 bp 
| style="width: 375px;" |
<s>T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC</s>

T7 AG    TAATACGACTCACTATAAGG

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

1610 bp

| style="width: 259px;" | 
|-
| 
| 
| style="width: 66px;" | 
| style="width: 13px;" | 
| 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|-
| Cre
| pT7AG-Cre 
| style="width: 66px;" | 
| style="width: 13px;" | 2942 
|  ?
| style="width: 40px;" | 
| style="width: 375px;" |
T7AG TAGTAATACGACTCACTATAAGG

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

1613 bp

| style="width: 259px;" | 
|-
| Fluc
| T7-VEE-FLuc 
| style="width: 66px;" | 
| style="width: 13px;" | 2889
| 35/F1 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|-
| Fluc
| pT7AG-Fluc-pA 
| style="width: 66px;" | 
| style="width: 13px;" | 2952 
| 35/F1 
| style="width: 40px;" | 
| style="width: 375px;" |
T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC

or T7AG TAATACGACTCACTATAAGG

+ VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

| style="width: 259px;" | 
|-
| RFP
|  T7-VEE-TagRFP 
| style="width: 66px;" | 
| style="width: 13px;" | 2904 
| 35/F5 
| style="width: 40px;" | 
| style="width: 375px;" | T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag,  
| style="width: 259px;" | 
|-
| 8000nt mRNA, self amplify TagGFP2 mRNA
| T7-VEE-TagGFP2-E3L-8000 
| style="width: 66px;" | 
| style="width: 13px;" | 2951 
| 33/I1 
| style="width: 40px;" | 8770
| style="width: 375px;" | Xba1, Mlu1, PacI
| style="width: 259px;" | for thermo project
|-
| EGFP mRNA
| T7-VEE-EGFP 
| style="width: 66px;" | EG-307, EG-320 
| style="width: 13px;" | 2885 
| 35/F7 
| style="width: 40px;" | 1093, 8642
| style="width: 375px;" |
PCR using 

T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag, 1093 bp PCR

product with pA and T7 promoter

| style="width: 259px;" |
not SAM, if by PCR

= == =

Digestion with Xba1,  Mlu1, PacI: make SAM EGFP mRNA

|-
| EGFP mRNA ARCA
| 
| style="width: 66px;" | 
| style="width: 13px;" | 
| 
| style="width: 40px;" | 
| style="width: 375px;" |
T7AG-HBG-F TAATACGACTCACTATAAGGtcttctggtccccacagac (not the best primer)

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

1149 bp

| style="width: 259px;" | 
|-
| 
| T7AG-EGFP 
| style="width: 66px;" | 
| style="width: 13px;" | 2953 
|  ?
| style="width: 40px;" | 
| style="width: 375px;" |
T7AG-HBG-F TAATACGACTCACTATAAGGtcttctggtccccacagac

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

1149bp

| style="width: 259px;" | 
|-
| 
| T7-VEE-TagGFP2 
| style="width: 66px;" | 
| style="width: 13px;" | 2926
|  ?
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|-
| FLP self amplifying  mRNA
| T7-VEE-NLS-Flp 
| style="width: 66px;" |
EG-1044

EG-1045

| style="width: 13px;" | 2895 
| 33/I3 
| style="width: 40px;" | 1724, 9283 
| style="width: 375px;" |
to make template by PCR  for EG-1044

<s>T7-HBG-F</s>

<s>TAATACGACTCACTATAAGGACATTTGCTTCTGACACAAC </s>

T7-NLS-FLPe

TAATACGACTCACTATAGGGATGGCCCCCAAGAAAAAGCGG

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

1724 bp

---

for EG-1045, digest plasmid with Xba1, Mlu1, PacI

| style="width: 259px;" | not SAM, if by PCR
Digestion with Xba1,  Mlu1, PacI: make SAM EGFP mRNA

|-
| FLuc  mRNA
| T7-VEE-FLuc 
| style="width: 66px;" | EG-316 
| style="width: 13px;" |
2889

2952

| 35/F1 
| style="width: 40px;" | 2023
| style="width: 375px;" |
to make template for Cleancap by PCR, superHiFi 1st round

2889-PCR-F:  tcttctggtccccacagactcagagagaacccGCCACCATGGAAGACGCCA

+ VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

2023 bp

2nd round, Taq

T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC

+ VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

digestion:

digest 2953 with Mlu1, purify DNA with glycogen + propanol

==

digest 2889 with Mlu1 for self amplify mRNA

| style="width: 259px;" | 
|-
| NanoFluc
| pRham-T7ag-NLuc-2A-EGFP 
| style="width: 66px;" | 
| style="width: 13px;" | 2935 
| 35/H3 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|-
| Rluc
| pT7ag-Rluc 
| style="width: 66px;" | 
| style="width: 13px;" | 2934 
| 35/H1 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|-
| Rluc
| T7ag-VEE-RLuc 
| style="width: 66px;" | 
| style="width: 13px;" | 2946 
| 35/H4 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|-
| 
| eSpCas9-T2A-EGFP 
| style="width: 66px;" | 
| style="width: 13px;" | 2931
| 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | can not use for mRNA production
|-
| Cas9-EGFP, self amplify mRNA
| T7-VEE-eSPCas9-EGFP 
| style="width: 66px;" |
EG-317,

EG-314

| style="width: 13px;" | 2938 
| 35/G9 
| style="width: 40px;" | 
| style="width: 375px;" |
for Cas9-EGFP SAM: digest 2938 with Mlu1

For  Cas9-EGFP, PCR using T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC + VEE-pA-R GAATTAATTCCCCTCGACGCGTTCTAG

| style="width: 259px;" |
EG-317: NLS-eCas9-T2A-EGFP Self-Amplifying mRNA, GSK or331295-1

EG-314: NLS-eCas9-T2A-EGFP mRNA (CleanCap), or331295-2

|-
| eCas9 self amplify mRNA
| T7ag-VEE-eCas9 
| style="width: 66px;" | EG-322 
| style="width: 13px;" | 2947 
| 35/H5 
| style="width: 40px;" | 
| style="width: 375px;" | digest 2947 with Mlu1, very low mRNA yield
| style="width: 259px;" | GSK or331295-4
|-
| eCas9 cleancap 
| T7ag-pA-eCas9 
| style="width: 66px;" | EG-321 
| style="width: 13px;" | 2939 
| 35/H2  
| style="width: 40px;" | 
| style="width: 375px;" | digest 2939 with Mlu1
| style="width: 259px;" | GSK r331295-3
|-
| 
| 
| style="width: 66px;" | 
| style="width: 13px;" | 
| 
| style="width: 40px;" | 
| style="width: 375px;" | 
| style="width: 259px;" | 
|}

= RNA Synthesis with Modified Nucleotides (E2050) =

'''Protocols.io also provides an [https://www.protocols.io/view/RNA-Synthesis-with-Modified-Nucleotides-E2050-cv7w9m https://www.protocols.io/view/RNA-Synthesis-with-Modified-Nucleotides-E2050-cv7w9m] interactive version of this protocol</a> where you can discover and share optimizations with the research community. '''

We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes. The kit is capable of synthesizing biotin- or dye-modified RNA with the following protocol. The recommended molar ratio of modified NTP (Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP) to standard NTP is 1:2. The following reaction set-up assumes modified UTP is used. Please note that Dye- or Biotin-NTPs are not supplied with the kit.

#Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes.
#Assemble the reaction at room temperature in the following order:

{|
|-
| Nuclease-free water 
| X µl
|-
| NTP Buffer Mix
| 5 µl (5 mM each NTP final)
|-
| Modified UTP (10 mM)
| 5 µl (2.5 mM final)
|-
| Template DNA
| X µl (1 µg)
|-
| T7 RNA Polymerase Mix
| 2 µl
|-
| Total reaction volume
| 20 µl 
|}

# Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours. For short (< 300 nt) transcripts incubate at 37°C for 4–16 hours. Note that the ratio of modified nucleotide to standard nucleotide can be adjusted by varying the amount of the NTP Buffer Mix and modified nucleotide. For complete modified nucleotide substitution we recommended using the T7 High Yield RNA Synthesis Kit  [https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit]  NEB E2040, in which the four nucleotides are supplied separately. Figure 1 shows the time course of labeled RNA synthesis using 1 µg control template with Biotin-16-UTP and Fluorescein-12-UTP following the above reaction setup. Modified ribonucleotides reduce transcription efficiency; therefore, lower transcription yields should be expected as compared to transcription using unmodified NTPs. In general, Biotin-NTP and Aminoallyl-NTP have an insignificant effect on yields, while lower yields can be expected for transcription reactions containing Fluorescein-NTP or Cy-NTP. In addition, transcripts containing modified ribonucleotides have reduced electrophoretic mobility due to higher molecular weight. [https://www.neb.com/~/media/Catalog/All-Protocols/494442EBCCE947D08CDFDFBEA28E8416/Content/E2050c.jpg&nbsp https://www.neb.com/%7E/media/Catalog/All-Protocols/494442EBCCE947D08CDFDFBEA28E8416/Content/E2050c.jpg&nbsp];
#'''Figure 1. RNA synthesis with modified nucleotides''' Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.
#''Optional:'' To remove template DNA, add 30 μl nuclease-free water and 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.
#Proceed with purification synthesized RNA (we recommend the Monarch RNA Cleanup Kits,  [https://www.neb.com/products/t2040-monarch-rna-cleanup-kit-50-ug https://www.neb.com/products/t2040-monarch-rna-cleanup-kit-50-ug] NEB T2040 or  [https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug] T2050 or analysis of transcription products by gel electrophoresis.

EGFP mRNA Protocol to make template for Cleancap by PCR

T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

T7-HBA2-F is not as good as T7AG-HBG-F

Template: 2885 T7-VEE-EGFP

(F primer T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC- for cleancap)

sequence, 1113 bp

GTTAATACGACTCACTATAag TCTTCTGGTCCCCACAGACTCAGAGAGAACCC Kozak gccacc

EGFP atggtgTCTaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa Not1 GCGGCCGC AAGCTGCTTACATAGAACTCGCGGCGATTG

Sph1

GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

PacI

ttaattaa

Xba1

tctaga

Mlu1

acgcgT CGAGGGGAATTAATTC

(R primer VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag )

T7-EGFP-F: TAATACGACTCACTATAAGGAGACCATGGTGAGCAAGGGC

Cleancap, mw 1145.60 g

1145.6 g in 1 L: 1M

1145.6 mg in 1 ml: 1M

11.45 mg in 1 ml: 10 uM

11.45 mg in 100 ul: 100uM 

1XIVT reaction buffer:

50 mM Tris-HCl, pH 7.5
15 mM MgCl2
5 mM dithiothreitol (DTT)
2 mM spermidine
Make 10X stock and store at -20°C.

10X NTPs
25 mM each of ATP, CTP, GTP, and UTP
Store at -20°C.

Inorganic pyrophosphatase

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate: P2O7-4 + H2O -> 2HPO4-2. Inorganic pyrophosphate is released when a nucleoside triphosphate is incorporated/polymerized into the growing chain. This helps to prevent against any inhibitory effect of having pyrophosphate around (i.e. prevents the "reverse" reaction.) This is an optional component of the transcription reaction. If you leave it out, often you will see something precipitate (white) in your transcription reaction. This is the pyrophosphate.

Make a 0.1 U/μL stock solution in H2O and store at -20°C.

Transcription reaction
For a 100 μL reaction ("preparative scale"):
10 μL 10X transcription buffer
10 μL 10X NTPs
xx μL DNA template (5–10 pmol)
5 μL inorganic pyrophosphatase (0.1 U/μL): 0.005 U/μL final concentration
xx μL T7 RNAP (25 U/μL final concentration)
1 μl RNase inhibitor (Murine RNase Inhibitor, NEB, 40000 U/ml)

================
Add the desired protospacer sequence to the T7FwdVar oligo and order the oligo from your favorite oligonucleotide supplier. There are many programs available for protospacer design that attempt to optimize on- and/or off-target activity. Which program is most useful depends upon many factors including type of editing, organism being edited, etc. Choice of protospacer design program is beyond the scope of this protocol.

The transcript will start with the bolded G just 5’ of the dashes in the T7FwdVar oligo. T7 RNA polymerase requires a 5’ G for proper transcript initiation. If your protospacer has a G at the 5’ end, you can omit it from the T7FwdVar design to avoid duplication of the G. If your protospacer has a C, T, or A at the 5’ end, add the whole protospacer sequence to T7FwdVar. In this case, there will be an extra G added to the 5’ end of the protospacer, but literature indicates this will have minimal effect unless your guide is very short.

Primers:
T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )
T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )
T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’)
T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )

For eachT7FwdVar oligo you designed, set up the following PCR (total volume should be 20.0 µL). Make sure everything is RNase free and filter tips are used. Furthermore, wipe down everything (in every step of protocol) with RNase Away to ensure no contamination with RNAse.

10.6 µl DEPC-treated H2O
4.0 uL 5x Phusion HF Buffer
0.4 µl 10 mM dNTPs
0.4 µl T7FwdVar (1 µM)
0.4 µl T7RevLong (1 µM)
2 µl T7FwdAmp (10 µM)
2 µl T7RevAmp (10 µM)
0.2 µl Phusion HF DNA polymerase (2u/µl)

If making multiple sgRNA templates, prepare a master mix with all components except T7FwdVar. Include a no template control (omit T7FwdVar).

Primers:
T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )
T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )
T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’)
T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )

Run PCR:
98˚ 30 sec
98˚ 10 sec
51˚ 10 sec
72˚ 10 sec
30x steps 2-4
72˚ 2 min
4˚ hold

No PCR cleanup necessary at this point

We like to use HiScribe T7 High Yield RNA Synthesis Kit but any T7 RNA synthesis kit should be fine.

Mix the following to make 20 µl totalT7 transcriptionmix
volume reagent
2 µl 10x Buffer 1x
2 µl "ATP (100 mM), 10 mM"
2 µl "GTP (100 mM), 10 mM"
2 µl "CTP (100 mM), 10 mM"
2 µl "UTP (100 mM), 10 mM"
8 µl "DNA template from Step 3, (usually ~4µg, 50 pmol)"
2 µl T7 RNA polymerase mix

Incubate transcription mix for ~18 hours (over night) at 37˚ in a thermocycler
18:00:00 START
37 °C

Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler
00:15:00 37C incubationSTART
DNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
37 °C

T7 in vitro transcription results in RNA carrying a 5'-triphosphate group. This triggers a RIG-I-mediated innate immnue response in mammalian cells and can cause cell death, particularly in primary cells. We highly recommend treating your IVT sgRNA with recombinant Shrimp Alkaline Phosphatase (rSAP) before proceeding to the purification step. We found that rSAP treatment must be rigorous to completely remove all 5-PPP groups from your RNA. However, rSAP binds tightly to RNA and NEB recommends to only use the minimal amount needed.

Add 3ul of NEB CutSmart Buffer (comes with the rSAP enzyme)
Add 4µl of H2O
Add 2ul (2 units) of rSAP

Shrimp Alkaline Phosphatase (rSAP) - 500 unitsNew England BiolabsCatalog #M0371S

Mix well and incubate at 37C for 3h
03:00:00 START
37 °C

sgRNAs need to be purfied before transfection. There are different methods one could purify their sgRNAs. We found that while SPRI bead clean-up of RNAs is quick and gives reliable yields (see older versions of protocol), SPRI bead purified sgRNAs still cause an elevated immune response even after Phosphatase treatment. We therefore tested different column purification kits and found that column purification of sgRNAs completely eliminates the immune response after Phosphatase treatment.

We use the Qiagen RNeasy Mini Prep Kit and follow the manufacturer's instructions with the following modifications:
Yields of from column purification can be low as the sgRNAs are small and most kits are designed to bind longer RNAs. We found the Qiagen RNeasy kit works ok, but others recommend the miRNeasy kit from Qiagen. It might be worth trying a few different kits to optimize your yield.

Adjust sgRNA sample to a volume of 100 µl with RNase-free water.
Add 350 µl RLT Buffer to the sample

Add 250 µl 100% ethanol and mix well by pipetting.

Tranfer the sample (~700 µl) to an RNeasy mini spin column; spin for 15 sec at >8000 g
Then transferr the remainder onto the same column; spin for 15 sec

Add 500 µl RPE Buffer; spin 15 sec
Repeated this wash step
Moved spin column to a new collection tube and spin for 1 min to dry the membrane completely

Move spin column to an RNAse-free 1.5 ml microfuge tube
Add 33 µl DEPC-treated H2O; spin 1 min
Optional: Repeat the elution to collect any remaining RNA

Measure your RNA concentration by Nanodrop or Qubit. You can also check integrity/corrrect size of the sgRNA(s) on the RNA tapestation. Store sgRNAs at -80C
https://www.protocols.io/run/in-vitro-transcription-of-guide-rnas-and-5-triphos-bqjbmuin

2959

2022-04-02T19:20:58Z

Paul.xu:

T7AG-Flp

subclone from 2895 to 2953 using Nde1-Mlu1

[[File:2959 2960 digestion.JPG|RTENOTITLE]]

TAATACGACTCACTATAAGGtcttctggtccccacagactcagagagaacccatatgtaaGGCGCGCCCACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAATTGCCACCATGGCCCCCAAGAAAAAGCGGAAAGTGATGAGCCAGTTCGACATCCTGTGCAAGACCCCCCCCAAGGTGCTCGTGCGGCAGTTCGTGGAAAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCTCTTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGCGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAAGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTTACCATCATCCCCTACAACGGCCAGAAGCACCAGAGCGACATCACCGATATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAAGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGAGATCACAGAGAAGATCCTGAACAGCTTCGAGTACACCAGCCGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACCTTTATCAACTGCGGCCGGTTCTCCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTCGTGACCGAGACAAAGACCAGCGTGTCCCGGCACATCTACTTTTTCAGCGCCAGAGGCCGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGAAACAGCGAGCCCGTGCTGAAGAGAGTGAACCGGACCGGCAACAGCAGCTCCAACAAGCAGGAATACCAGCTGCTGAAGGACAACCTCGTGCGGTCCTACAACAAGGCCCTGAAGAAAAACGCCCCCTACCCCATCTTCGCCATTAAGAACGGCCCCAAGTCCCACATCGGCCGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACAGAGCTGACCAACGTCGTGGGCAATTGGAGCGACAAGAGGGCCTCTGCCGTGGCCAGAACCACCTACACCCACCAGATCACAGCCATCCCCGACCACTACTTCGCCCTGGTGTCTCGGTACTACGCCTACGACCCCATCAGCAAAGAGATGATCGCCCTGAAGGACGAGACAAACCCCATCGAGGAATGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCTGCCTGGAACGGCATCATCTCCCAGGAAGTGCTGGACTACCTGAGCAGCTACATCAACCGGCGGATCTAGCGGCCGCTTAAGCTGCTTACCGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCCGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC
<pre>>A220401-011_M22_1FC0DAA006_premix.ab1 1231 4/2/2022
2959 T7AG-Flp
sequencing using T7AG

GNNNNNNNNANNCNNNTNNNNGATGAGACNTCTTNANATATTATTGTCAGAGCTCCCTGTAGGTGCTTTAGCGGGACGCTATGTCCGGATTCTGATGTAC
CTGCTGAGGTAATATAGCCTTTTGTGGGAGATAATTGAGTTACATGTGCGGGATGAGATGCTGCCCTCGGCCCTGCCCTTCCGGGCTCCTTCNCTGCCAT
TCCTCCATGGTCTTTGTCTCGCTGATCTTCGACATCATGTATTTGATGATGGGGTTCAAGGACAAGACCCAAAACGCCACCATCCTGNAAGGGTCGGGAA
TGGNTGGATCCTGGCGTGGGAGTTTGCCTTCGTCCCCTACAACGGCCCTATTGTCCAGAGCGTTGTCACCGATATCGTGTCCAGCCTGCAGCTGCAGTTC
GAGAGCAGCGAGGAAGCCCACGAGGGCAACAGCCACAGCAAGAAGATGCT
GAATGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGATATCACAGAGAAGA
TCCTGAACAGCTTCGAGTACACCAGCCGGTTCACCAAGACCAAGACCCTG
TACCAGTTCCTGTTCCTGGCCACCTTTATCAACTGCGGCCGGTTCTCCNA
CATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACC
TGGGCGTGATCATTCAGTGNCTCGTGACCGANACATAGACCAGCGTGTCC
CGGCNCATCTACTTTTTCAGCGCCAGANGNCGGATCGACCCCCTGGTGTA
CCTGGNCNAGTTCCTGAGAACAGCGAGCCCGTGCTGAANAGAGTGAACCG
GACCGGCAACAGCANCTGCNACAAGCANGANTACCAGCTGCNTGANGGAC
NATCTCNTGNNGGTCTNTACANNATGTCTCNTGANNAAAACGCCCCACTN
ACNCNTCTTCGCCATTAANANGGCCCAAGTCCNNACATCGGCGGCACTGA
TGACCAGNTTTCNNAGCATGANNGNTGANNAGCTGACNNCGTCNTGGGTC
AATTGNNTCNACANANNNNTNTNNNTNNNNNANNNTNNNNNNCNNATNNN
NGCATCNCGACNNCTACTTNNCNTGNGNNNCNGNACTACNCNACGACCNT
NNGNNNNNNNGATNNNNNAANNNNANNNANNNNNTNNGGATGNAGCNANT
NGNNNNTNANNNNGNCNNNNGNATNNNNNNNNCNNNNGNNNNNNNNNNNT
NTNNNTNNNNNNCNNNNNANNNNNCNNNNNN

Alignment statistics for match #1
Score Expect Identities Gaps Strand
920 bits(498) 0.0 642/726(88%) 15/726(2%) Plus/Plus
Query 254 GTTCAAGGACAAGACCCAAAACGCCACCATCCTGNAAG-GGTCGGGAATG-GNTGGATCC 311
||||||| |||||||||| || |||||||||||| ||| | ||| | | |||||
Sbjct 383 GTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAAGCCAGCCTGAAGAAGCT-GATCC 441

Query 312 TGGCGTGGGAGTTTGCCTTCGTCCCCTACAACGGCCCTATTGTCCAGAGCGTTGTCACCG 371
|| ||||||||| || || ||||||||||||||| | |||||||| ||||||
Sbjct 442 CCGCCTGGGAGTTTACCATCATCCCCTACAACGGCCAGAAGCACCAGAGCGACATCACCG 501

Query 372 ATATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAAGCCCACGAGGGCAACA 431
||||||||||||||||||||||||||||||||||||||||||||||| || |||||||||
Sbjct 502 ATATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAAGCCGACAAGGGCAACA 561

Query 432 GCCACAGCAAGAAGATGCTGAATGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGATATCA 491
|||||||||||||||||||||| |||||||||||||||||||||||||||||||| ||||
Sbjct 562 GCCACAGCAAGAAGATGCTGAAGGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGAGATCA 621

Query 492 CAGAGAAGATCCTGAACAGCTTCGAGTACACCAGCCGGTTCACCAAGACCAAGACCCTGT 551
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 622 CAGAGAAGATCCTGAACAGCTTCGAGTACACCAGCCGGTTCACCAAGACCAAGACCCTGT 681

Query 552 ACCAGTTCCTGTTCCTGGCCACCTTTATCAACTGCGGCCGGTTCTCCNACATCAAGAACG 611
||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||
Sbjct 682 ACCAGTTCCTGTTCCTGGCCACCTTTATCAACTGCGGCCGGTTCTCCGACATCAAGAACG 741

Query 612 TGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGNC 671
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |
Sbjct 742 TGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCC 801

Query 672 TCGTGACCGANACATAGACCAGCGTGTCCCGGCNCATCTACTTTTTCAGCGCCAGANGNC 731
|||||||||| ||| |||||||||||||||||| |||||||||||||||||||||| | |
Sbjct 802 TCGTGACCGAGACAAAGACCAGCGTGTCCCGGCACATCTACTTTTTCAGCGCCAGAGGCC 861

Query 732 GGATCGACCCCCTGGTGTACCTGGNCNAGTTCCTGAG-AACAGCGAGCCCGTGCTGAANA 790
|||||||||||||||||||||||| | |||||||||| |||||||||||||||||||| |
Sbjct 862 GGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGAAACAGCGAGCCCGTGCTGAAGA 921

Query 791 GAGTGAACCGGACCGGCAACAGCANCTGCNACAAGCANGANTACCAGCTGCNTGANGGAC 850
|||||||||||||||||||||||| || | ||||||| || |||||||||| ||| ||||
Sbjct 922 GAGTGAACCGGACCGGCAACAGCAGCTCCAACAAGCAGGAATACCAGCTGC-TGAAGGAC 980

Query 851 NATCTCNTGNNGGTCTNTACANNATGTCTCNTGANNAAAA-CGCCCCACTNACNCNTCTT 909
| ||| || |||| |||| | | | | ||| |||| |||||| || | | ||||
Sbjct 981 AACCTCGTGC-GGTCC-TACAACAAGGCCC-TGAAGAAAAACGCCCC-CTACCCCATCTT 1036

Query 910 CGCCATTAANAN-GGCCC-AAGTCCNNACATCGGC-GGCA-CTGATGACCAGNTTTCNNA 965
||||||||| | ||||| |||||| |||||||| |||| ||||||||||| |||| |
Sbjct 1037 CGCCATTAAGAACGGCCCCAAGTCCC-ACATCGGCCGGCACCTGATGACCAGCTTTCTGA 1095

Query 966 GCATGA 971
||||||
Sbjct 1096 GCATGA 1101

</pre>
[[Category:Cloning]]

2960

2022-04-02T18:57:11Z

Paul.xu:

T7AG-eCas9-EGFP

cloning using Mlu1-Nde1 from 2938 to 2953

TAATACGACTCACTATAAGGtcttctggtccccacagactcagagagaaccCATATGCTAGCtagagaataaactagtattcttctggtccccacagactcagagagaaccGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGGCCGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGCGGCCGCTTAAGCTGCTTACCGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCCGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

<pre>
Score Expect Identities Gaps Strand
1568 bits(849) 0.0 863/875(99%) 1/875(0%) Plus/Plus
Query 20 ACCCATATGCTAGCTAGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGA 79
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 49 ACCCATATGCTAGCTAGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGA 108

Query 80 ACCGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTAC 139
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 109 ACCGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTAC 168

Query 140 AAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTC 199
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 169 AAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTC 228

Query 200 CCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGG 259
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 229 CCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGG 288

Query 260 GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACC 319
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 289 GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACC 348

Query 320 GACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACA 379
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 349 GACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACA 408

Query 380 GCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGG 439
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 409 GCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGG 468

Query 440 ATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTC 499
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 469 ATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTC 528

Query 500 CACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATC 559
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 529 CACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATC 588

Query 560 TTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTG 619
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 589 TTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTG 648

Query 620 AGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTG 679
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 649 AGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTG 708

Query 680 GCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC 739
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 709 GCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC 768

Query 740 AGCGACGTGGANNAGCTGTTCATCCAGCTGGTGCAGACCTACNACCAGCTGNTCGAGGAA 799
||||||||||| ||||||||||||||||||||||||||||| |||||||| ||||||||
Sbjct 769 AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAA 828

Query 800 AACCCCATCAACGCCAGCGGCGTNGNCGCCNAGGCCATCCTGNCTGCCANACTGAGCAAG 859
||||||||||||||||||||||| | |||| ||||||||||| |||||| ||||||||||
Sbjct 829 AACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAG 888

Query 860 AGCAGACGGCTGG-AAATCNGATCGNCCAGCTGCC 893
||||||||||||| ||||| ||||| |||||||||
Sbjct 889 AGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCC 923

>A220401-011_K22_1FC0DAA007_premix.ab1 1180 4/2/2022
seq T7AG Promoter
NNNNNNNNNNNTCNGANNNACCCATATGCTAGCTAGAGAATAAACTAGTA
TTCTTCTGGTCCCCACAGACTCAGAGAGAACCGCCACCATGGACTATAAG
GACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGA
CGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCC
CAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCT
GTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATT
CAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCG
GAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAG
AGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCT
GCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC
ACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGG
CACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTA
CCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGG
CCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGG
GGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGA
NNAGCTGTTCATCCAGCTGGTGCAGACCTACNACCAGCTGNTCGAGGAAA
ACCCCATCAACGCCAGCGGCGTNGNCGCCNAGGCCATCCTGNCTGCCANA
CTGAGCAAGAGCAGACGGCTGGAAATCNGATCGNCCAGCTGCCNNNNNGA
AGAAGATGCCTGNNNNNAACNGATTGCCCTGAGCTGGNNTGACCCCNACT
CANANCACTCGACTNNNAGNNGCNNCTGCAGCTGANNGNCNCTACGACGA
CGACTGNNACNGCTGGNNNATCGNNACANTACNCNANCTGNTCTGGCNNN
NNNNGNCNANCNTCNGNTNNNNNNNNTGNANNNNNNNNNTNNNNNNNNNA
GNCCNNNNANNNNNNNNNNNNNNGNCNGANNGNNNNNNNNNNNNNNNANN
NNNNNNNGCNNNNNNNNNNCCNNNNNNNNG
</pre>

[[Category:Cloning]]

File:2959 2960 digestion.JPG

2022-04-01T23:28:55Z

Paul.xu:

2892

2022-03-27T12:44:54Z

Paul.xu: Created page with "T7-VEE-eCas9-T2A-EGFP not done yet"

T7-VEE-eCas9-T2A-EGFP
not done yet

2931

2022-03-27T11:53:15Z

Paul.xu: Created page with "eSpCas9-T2A-EGFP <pre>U6 promoter gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggta..."

eSpCas9-T2A-EGFP
<pre>U6 promoter

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac
gaaacaccg
AAVs1 (AS) (PPP1R12C)

ggggccactagggacaggat

gRNA Scaffold
gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc

ttttttgttttagagctagaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggc
Xba1-Kpn1
tctagaggtacc

tttagcgcgtgcgccaattctgcagacaaatggctctagaggtacccgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccgggggggggggggggggggggggggggggggggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttggaccggt
Kozak
gccacc
3X Flag SV40NLS
atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagcc
Cas9
gacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaag

gaattcggcagtgga
T2A
gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggccca
EGFP
gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaaggaattctaa

ctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctgggga

Not1
gcggccgc
aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt
</pre>

2938

2022-03-27T11:38:59Z

Paul.xu:

T7-VEE-eSPCas9-EGFP

PCR 2931-->2893

[[File:2935 2938 2939 2942.JPG|RTENOTITLE]]

<pre>Gibson mix cloning
PCR from 2931
2935-F4
TCTAGTCCGCCAAGTCTAGcatatgCTAGCtagagaataaac

1uM 2935-F1
atcatatgCTAGCtagagaataaactagtattcttctggtccccacagactc
1 uM 2938-F1
cttctggtccccacagactcagagagaaccGCCACCATGGACTATAAGGAC

2938-R1
TTCTATGTAAGCAGCTTGCGGCCGCTCCCCAGCATGCCTG
(CAGGCATGCTGGGGAGCGGCCGCAAGCTGCTTACATAGAA)
====
Use Nde1-AflII for cloning into 2895 or 2926, not for gibson mix unless using additional PCR with VEE-GM-F +
VEE-GM-R
2938-R2
TTCTATGTAAGCAGCTTaAGCGGCCGCTCCCCAGCATGC
(GCATGCTGGGGAGCGGCCGCTtAAGCTGCTTACATAGAA)

===
VEE-GM-F

CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGTCTAG

VEE-GM-R
GGCATGCCAATCGCCGCGAGTTCTATGTAAGCAGCTTGCG
(CGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCC)

===

Enzyme-free cloning
PCR from vector 2893

EFC-V-F CTCGAGGATGGGGACTAGtg
EFC-V-R (2942-R)
TGAGTCGTATTAagcttcatatGAGCTCGCGTTGCTGGCG
(CGCCAGCAACGCGAGCTCatatgaagctTAATACGACTCA)

PCR insert from 2931
EFC-F CatatgaagctTAATACGACTCACTATAagGagaataaactagtattcttc
1uM 2935-F1
atcatatgCTAGCtagagaataaactagtattcttctggtccccacagactc
1 uM 2938-F1
cttctggtccccacagactcagagagaaccGCCACCATGGACTATAAGGAC
<strike>
EFC-F1 CatatgaagctTAATACGACTCACTATAagGagaataaac
EFC- F2 TCACTATAagGagaataaac tagtattcttctggtcccca
</strike>

2938-EFC-R
caCTAGTCCCCATCCTCGAGCGGCCGCTCCCCAGCATG
caCTAGTCCCCATCCTCGAGCGGCCGCTCCCCAGCATGC
(GCATGCTGGGGAGCGGCCGCTCGAGGATGGGGACTAGtg)

mix both PCR, digest with DpnI, purify and transform 10G

TAATACGACTCACTATAGATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAGGCAGACGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATTCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGTCTAG
Nde1
catatgCTAGCtagagaataaactagtattcttctggtccccacagactcagagagaacc
Kozak
GCCACC
eCas9
ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGGCCGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA

EGFP
GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGA
GAATAGCAGGCATGCTGGGGA
Not1
GCGGCCGC

TTAAGCTGCTTACCGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCCGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

====

>210317-21_C07_1FC0DAA034.ab1 1191, 3/18/2021
2938 in Vee vector, seq with VEE-seq-R
NNNNNNNTNNNNNGCNNNTNGCGGCCGCTCCCCAGCATGCCTGCTATTCT
CTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATA
GAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGA
AAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGG
GGCAAACAACAGATGGCTGGCAACTAGAAGGCACAGTCGAGGCTGATCAG
CGAGCTCTAGTTAGAATTCCTTGTACAGCTCGTCCATGCCGAGAGTGATC
CCGGCGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTT
GGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCA
GCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCG
AGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTT
GATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGT
AGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCG
ATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCAC
CTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCT
CCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTC
ATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGT
CACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACT
TCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACG
CTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCAC
CACCCCGGTGAACAGCTCCTCGCCCTTGCTCACTGGGCCAGGATTCTCCT
CGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCTCCACTGCCGAAT
TCCTTTTTCTTTTTTGCCTGGCCGGCCTTTTTCGTGGCCGCCGGCCTTTT
GTCGCCTCCCAGCTGANACAGGTCGATCCGGGTCTCGTACAGGCCGGTGA
TGCTCTGGGGGATCAGGGNGGCGTCCAGCACCTCTTTGGGG

rev comp

CCCCAAAGAGGTGCTGGACGCCNCCCTGATCCCCCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGTNTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGCGGCCGCNANNNGCNNNNNANNNNNNN

Alignment statistics for match #1
Score Expect Identities Gaps Strand
2141 bits(1159) 0.0 1165/1169(99%) 0/1169(0%) Plus/Plus
Query 3 CCAAAGAGGTGCTGGACGCCNCCCTGATCCCCCAGAGCATCACCGGCCTGTACGAGACCC 62
|||||||||||||||||||| ||||||||| ||||||||||||||||||||||||||| |
Sbjct 4199 CCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACAC 4258

Query 63 GGATCGACCTGTNTCAGCTGGGAGGCGACaaaaggccggcggccacgaaaaaggccggcc 122
|||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4259 GGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCC 4318

Query 123 aggcaaaaaagaaaaaGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCG 182
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4319 AGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCG 4378

Query 183 GTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG 242
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4379 GTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG 4438

Query 243 TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG 302
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4439 TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG 4498

Query 303 AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA 362
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4499 AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA 4558

Query 363 AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCA 422
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4559 AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCA 4618

Query 423 GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCT 482
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4619 GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCT 4678

Query 483 ACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG 542
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4679 ACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG 4738

Query 543 TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG 602
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4739 TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG 4798

Query 603 AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA 662
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4799 AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA 4858

Query 663 TCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG 722
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4859 TCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG 4918

Query 723 AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC 782
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4919 AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC 4978

Query 783 CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA 842
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4979 CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA 5038

Query 843 ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG 902
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5039 ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG 5098

Query 903 GCATGGACGAGCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCC 962
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5099 GCATGGACGAGCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCC 5158

Query 963 TTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG 1022
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5159 TTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG 5218

Query 1023 TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG 1082
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5219 TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG 5278

Query 1083 GTGTCATTCTATTCTggggggtggggtggggCAGGACAGCAAGGGGGAGGATTGGGAAGA 1142
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5279 GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGA 5338

Query 1143 GAATAGCAGGCATGCTGGGGAGCGGCCGC 1171
|||||||||||||||||||||||||||||
Sbjct 5339 GAATAGCAGGCATGCTGGGGAGCGGCCGC 5367

>210317-21_A07_1FC0DAA035.ab1 1175 3/18/2021
2938 in Vee vector, seq with VEE-seq-F
good
NNNNNNNNNTCTNCGGCTANCTGATGGACTACGACATAGTCTAGTCCGCC
AAGTCTAGCATATGCTAGCTAGAGAATAAACTAGTATTCTTCTGGTCCCC
ACAGACTCAGAGAGAACCGCCACCATGGACTATAAGGACCACGACGGAGA
CTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCC
CAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAG
AAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGT
GATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCA
ACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTC
GACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG
AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCA
GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAG
TCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGG
CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACC
ACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTG
ATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGAT
CGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCC
AGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCC
AGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAG
ACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCC
TGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAG
AGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACAC
CTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG
CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGC
GACATCCTGAGAGTGAANNCCGAGA

Score Expect Identities Gaps Strand
2056 bits(1113) 0.0 1115/1117(99%) 0/1117(0%) Plus/Plus
Query 59 CATATGCTAGCTAGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACC 118
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1 CATATGCTAGCTAGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACC 60

Query 119 GCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAA 178
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 61 GCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAA 120

Query 179 GACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCA 238
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121 GACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCA 180

Query 239 GCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCC 298
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 181 GCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCC 240

Query 299 GTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGAC 358
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 241 GTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGAC 300

Query 359 CGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC 418
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 301 CGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC 360

Query 419 GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATC 478
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 361 GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATC 420

Query 479 TGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCAC 538
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 421 TGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCAC 480

Query 539 AGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTC 598
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 481 AGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTC 540

Query 599 GGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGA 658
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 541 GGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGA 600

Query 659 AAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC 718
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 601 AAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC 660

Query 719 CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGC 778
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 661 CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGC 720

Query 779 GACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAAC 838
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 721 GACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAAC 780

Query 839 CCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGC 898
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 781 CCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGC 840

Query 899 AGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGA 958
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 841 AGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGA 900

Query 959 AACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCC 1018
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 901 AACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCC 960

Query 1019 GAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTG 1078
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 961 GAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTG 1020

Query 1079 GCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCC 1138
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1021 GCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCC 1080

Query 1139 ATCCTGCTGAGCGACATCCTGAGAGTGAANNCCGAGA 1175
||||||||||||||||||||||||||||| ||||||
Sbjct 1081 ATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGA 1117

</pre>
[[Category:Cloning]]

2947

2022-03-26T21:43:56Z

Paul.xu: Created page with "2947 T7ag-VEE-eCas9 subclone from 2939 to VEE vector Nde1-Not1, buffer3.1 <pre> TAATACGACTCACTATAGATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAG..."

2947 T7ag-VEE-eCas9
subclone from 2939 to VEE vector

Nde1-Not1, buffer3.1

<pre>
TAATACGACTCACTATAGATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAGGCAGACGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATTCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGTCTAG

Nde1 HindIII
catatgaagct
( T7 ag)
TAATACGACTCACTATA agG agaataaactagtattcttc tggtccccacagactcagagagaacc
GCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGGCCGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAA

TAG

Xho1
CTCGAGGATGGGGAC
TAGtgA

Not1
GCGGCCGC

GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

</pre>

2939

2022-03-26T19:27:40Z

Paul.xu:

<pre>not working--->
PCR from 2931
Gibson mix cloning

2935-F4
TCTAGTCCGCCAAGTCTAGcatatgCTAGCtagagaataaac

1uM 2935-F1
atcatatgCTAGCtagagaataaactagtattcttctggtccccacagactc
1 uM 2938-F1
cttctggtccccacagactcagagagaaccGCCACCATGGACTATAAGGAC
2939-R1
CTATGTAAGCAGCTTGCGGCCGCTATTCCTTTTTCTTTTTTGCCTG
(CAGGCAAAAAAGAAAAAGGAATAGCGGCCGCAAGCTGCTTACATAG)

========
Use Nde1-AflII for cloning into 2895 or 2926
2939-R2
TGTAAGCAGCTTaAGCTATTCCTTTTTCTTTTTTGCCTGG
(CCAGGCAAAAAAGAAAAAGGAATAGCTtAAGCTGCTTACA)

===
Enzyme-free cloning

<s>1uM 2935-F1
atcatatgCTAGCtagagaataaactagtattcttctggtccccacagactc</s>

<s>EFC-F CatatgaagctTAATACGACTCACTATAagGagaataaactagtattcttc </s>

PCR insert from 2931
EFC-F1 CatatgaagctTAATACGACTCACTATAagGagaataaac
EFC- F2 TCACTATAagGagaataaac tagtattcttctggtcccca

1 uM 2938-F1
cttctggtccccacagactcagagagaaccGCCACCATGGACTATAAGGAC

2939-EFC-R caCTAGTCCCCATCCTCGAGCTATTCCTTTTTCTTTTTTG
(CAAAAAAGAAAAAGGAATAGCTCGAGGATGGGGACTAGtg)

PCR from vector 2893

EFC-V-F CTCGAGGATGGGGACTAGtg
EFC-V-R (2942-R)
TGAGTCGTATTAagcttcatatGAGCTCGCGTTGCTGGCG
(CGCCAGCAACGCGAGCTCatatgaagctTAATACGACTCA)

digest with DpnI, purify, Gibson mix and transform 10G

</pre>

[[File:2935 2938 2939 2942.JPG|RTENOTITLE]] T7ag-pA-eCas9
<pre>Nde1 HindIII
catatgaagct
( T7 ag)
TAATACGACTCACTATA agG agaataaactagtattcttc tggtccccacagactcagagagaacc
GCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGGCCGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAA

TAG

Xho1
CTCGAGGATGGGGAC
TAGtgA

GCCGGCC
Not1
GCGGCCGC

GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATT
AAGCTGCTTACATAGAACTCGCGGCGATTG

Sph1
GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaa

Xba1
tctaga
acgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

>210316-33_A11_1FC0DAA038.ab1 1225 3/17/21
2939 seq using VEE-seq-R
NNNNNNNNNNNNNNGNNGCTTNTGNNTAAATGTTTTTTATTAGGCAGAAT
CCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTT
AGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCGC
GGCCGCGGCCGGCTCACTAGTCCCCATCCTCGAGCTATTCCTTTTTCTTT
TTTGCCTGGCCGGCCTTTTTCGTGGCCGCCGGCCTTTTGTCGCCTCCCAG
CTGAGACAGGTCGATCCGTGTCTCGTACAGGCCGGTGATGCTCTGGTGGA
TCAGGGTGGCGTCCAGCACCTCTTTGGTGCTGGTGTACCTCTTCCGGTCG
ATGGTGGTGTCAAAGTACTTGAAGGCGGCAGGGGCTCCCAGATTGGTCAG
GGTAAACAGGTGGATGATATTCTCGGCCTGCTCTCTGATGGGCTTATCCC
GGTGCTTGTTGTAGGCGGACAGCACTTTGTCCAGATTAGCGTCGGCCAGG
ATCACTCTCTTGGAGAACTCGCTGATCTGCTCGATGATCTCGTCCAGGTA
GTGCTTGTGCTGTTCCACAAACAGCTGTTTCTGCTCATTATCCTCGGGGG
AGCCCTTCAGCTTCTCATAGTGGCTGGCCAGGTACAGGAAGTTCACATAT
TTGGAGGGCAGGGCCAGTTCGTTTCCCTTCTGCAGTTCGCCGGCAGAGGC
CAGCATTCTCTTCCGGCCGTTTTCCAGCTCGAACAGGGAGTACTTAGGCA
GCTTGATGATCAGGTCCTTTTTCACTTCTTTGTAGCCCTTGGCTTCCAGA
AAGTCGATGGGATTCTTCTCGAAGCTGCTTCTTTCCATGATGGTGATCCC
CAGCAGCTCTTTCACACTCTTCAGTTTCTTGGACTTGCCCTTTTCCACTT
TGGCCACCACCAGCACAGAATAGGCCACGGTGGGGCTGTCGAAGCCGCCG
TACTTCTTAGGGTCCCAGTCCTTCTTTCTGGCGATCAGCTTATCGCTGTT
CCTCTTGGGCAGGATAGACTCTTTGCTGAAGCCGCCTGTCTGCACCTCGG
TCTTTTTCACGATATTCACTTGGGGCATGCTCAGCACTTTCCGCACGGTG
GCAAAATCCCGGCCCTTATCCCACACGATCTCCCCGGTTTCGCCGTTTGT
CTCGATCAGAGGCGCCTTCCGGATCTCGCCGTTGGCCAGGGTAATCTCGG
TCTTGAAAAAGTTCATGAAGTTGCC

rev comp
GGCAACTTCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATAGCTCGAGGATGGGGACTAGTGAGCCGGCCGCGGCCGCGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTANNCANAAGCNNCNNNNNNNNNNNNNN

Score Expect Identities Gaps Strand
2207 bits(1195) 0.0 1197/1198(99%) 0/1198(0%) Plus/Plus
Query 2 GCAACTTCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGG 61
||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3302 GCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGG 3361

Query 62 CGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATT 121
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3362 CGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATT 3421

Query 122 TTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGG 181
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3422 TTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGG 3481

Query 182 TGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGA 241
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3482 TGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGA 3541

Query 242 TCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGG 301
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3542 TCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGG 3601

Query 302 CCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTG 361
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3602 CCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTG 3661

Query 362 TGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCG 421
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3662 TGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCG 3721

Query 422 ACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTA 481
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3722 ACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTA 3781

Query 482 AGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAAC 541
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3782 AGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAAC 3841

Query 542 TGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCA 601
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3842 TGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCA 3901

Query 602 GCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGG 661
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3902 GCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGG 3961

Query 662 AACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAG 721
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3962 AACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAG 4021

Query 722 TGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATA 781
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4022 TGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATA 4081

Query 782 AGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAG 841
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4082 AGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAG 4141

Query 842 CCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCA 901
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4142 CCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCA 4201

Query 902 AAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGA 961
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4202 AAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGA 4261

Query 962 TCGACCTGTCTCAGCTGGGAGGCGACaaaaggccggcggccacgaaaaaggccggccagg 1021
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4262 TCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGG 4321

Query 1022 caaaaaagaaaaaGGAATAGCTCGAGGATGGGGACTAGTGAGCCGGCCGCGGCCGCGCTC 1081
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4322 CAAAAAAGAAAAAGGAATAGCTCGAGGATGGGGACTAGTGAGCCGGCCGCGGCCGCGCTC 4381

Query 1082 GCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAA 1141
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4382 GCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAA 4441

Query 1142 CTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTA 1199
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 4442 CTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTA 4499

>210316-33_G11_1FC0DAA039.ab1 1303 3/17/21
2939 seq using EFC-F1 CatatgaagctTAATACGACTCACTATAagGagaataaac
good

NNNNTNNNNNNNNNNNACTAGTATTCTTCTGGTCCCCACAGACTCAGAGA
GAACCGCCACCATGGACTATAaGGACCACGACGGAGACTACAAGGATCAT
GATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCG
GAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCG
GCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAG
TACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCA
CAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAA
CAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGA
CGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGC
CAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGG
AAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGAC
GAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAA
ACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCC
TGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTG
AACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC
CTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACG
CCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAAT
CTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCT
GATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACC
TGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGAC
CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCT
GGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAAAG
TGAACACCGAGATCACCAGGGCCCCCCTGAACGCCTCTATGATCAGNNAT
ACCAACAAGCACCACCAGGACCTGACCCTGCTGAAAGNNCTCGNGCGGAA
ACNNNTGCCTGAGAANNNCAANNGATTTTCTTNCANNNAANNAGAAACGG
CNACCCCGGTTNCTTNNNNNGNGGNCCNNCCNGAAAAATTCTCNNTNNNN
TGC

Score Expect Identities Gaps Strand
2132 bits(1154) 0.0 1182/1200(99%) 2/1200(0%) Plus/Plus
Query 17 ACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCGCCACCATGGACTATAaGGAC 76
||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||
Sbjct 22 ACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCGCCACCATGGACTATAAGGAC 81

Query 77 CACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCC 136
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 82 CACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCC 141

Query 137 CCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGC 196
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 142 CCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGC 201

Query 197 ATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAG 256
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 202 ATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAG 261

Query 257 GTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC 316
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 262 GTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC 321

Query 317 CTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGA 376
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 322 CTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGA 381

Query 377 ACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTC 436
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 382 ACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTC 441

Query 437 AGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTG 496
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 442 AGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTG 501

Query 497 GTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTG 556
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 502 GTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTG 561

Query 557 GCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACC 616
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 562 GCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACC 621

Query 617 GACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGC 676
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 622 GACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGC 681

Query 677 CACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATC 736
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 682 CACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATC 741

Query 737 CAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTG 796
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 742 CAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTG 801

Query 797 GACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATC 856
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 802 GACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATC 861

Query 857 GCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTG 916
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 862 GCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTG 921

Query 917 GGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTG 976
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 922 GGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTG 981

Query 977 AGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC 1036
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 982 AGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC 1041

Query 1037 GCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTG 1096
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1042 GCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTG 1101

Query 1097 AAAGTGAACACCGAGATCACCAGGGCCCCCCTGAACGCCTCTATGATCA-GNNATACCAA 1155
| |||||||||||||||||||| ||||||||||| |||||||||||||| | |||| |
Sbjct 1102 AGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACG-A 1160

Query 1156 CAAGCACCACCAGGACCTGACCCTGCTGAAAGNNCTCGNGCGGAAACNNNTGCCTGAGAA 1215
| |||||||||||||||||||||||||||||| |||| |||| | | ||||||||||
Sbjct 1161 CGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAA 1220

</pre>

[[Category:Cloning]]

2889

2022-03-26T13:57:06Z

Paul.xu: Created page with "T7-VEE-FLuc <pre>2889-F: TCATATGTAAGGCGCGCCACCATGGAAGACGCCAAAAACA 2889-R: ttGCGGCCGCGGCCGGCTTACAATTTGGACTTTCCG (CGGAAAGTCCAAATTGTAAGCCGGCCGCGGCCGCaa) clone into pMiniT, then s..."

T7-VEE-FLuc
<pre>2889-F:
TCATATGTAAGGCGCGCCACCATGGAAGACGCCAAAAACA
2889-R:
ttGCGGCCGCGGCCGGCTTACAATTTGGACTTTCCG
(CGGAAAGTCCAAATTGTAAGCCGGCCGCGGCCGCaa)
clone into pMiniT, then subclone using Nde1-Not1

>201228-13_O07_1F85DAA023.ab1 1192, 12/29/2020
seq with pminiT-F
NNNNNNNTNNNNNNNCGACCGATTGTTAGGTAATCGTCACCTGCAGGAAG
GTTTAAACGCATTTAGGTGACACTATAGAAGTGTGTATCGCTCGAGGGAT
CCGAATTCAGGAGGTAAAAACCATGATGCGGCCGCGGCCGGCTTACAATT
TGGACTTTCCGCCCTTCTTGGCCTTTATGAGGATCTCTCTGATTTTTCTT
GCGTCGAGTTTTCCGGTAAGACCTTTCGGTACTTCGTCCACAAACACAAC
TCCTCCGCGCAACTTTTTCGCGGTTGTTACTTGACTGGCGACGTAATCCA
CGATCTCTTTTTCCGTCATCGTCTTTCCGTGCTCCAAAACAACAACGGCG
GCGGGAAGTTCACCGGCGTCATCGTCGGGAAGACCTGCCACGCCCGCGTC
GAAGATGTTGGGGTGTTGTAACAATATCGATTCCAATTCAGCGGGGGCCA
CCTGATATCCTTTGTATTTAATTAAAGACTTCAAGCGGTCAACTATGAAG
AAGTGTTCGTCTTCGTCCCAGTAAGCTATGTCTCCAGAATGTAGCCATCC
ATCCTTGTCAATCAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGG
ACATAATCATAGGTCCTCTGACACATAATTCGCCTCTCTGATTAACGCCC
AGCGTTTTCCCGGTATCCAGATCCACAACCTTCGCTTCAAAAAATGGAAC
AACTTTACCGACCGCGCCCGGTTTATCATCCCCCTCGGGTGTAATCAGAA
TAGCTGATGTAGTCTCAGTGAGCCCATATCCTTGTCGTATCCCTGGAAGA
TGGAAGCGTTTTGCAACCGCTTCCCCGACTTCTTTCGAAAGAGGTGCGCC
CCCAGAAGCAATTTCGTGTAAATTAGATAAATCGTATTTGTCAATCAGAG
TGCTTTTGGCGAAGAATGAAAATAGGGTTGGTACTAGCAACGCACTTTGA
ATTTTGTAATCCTGAAGGGATCGTAAAAACAGCTCTTCTTCAAATCTATA
CATTAAGACGACTCGAAATCCACATATCAAATATCCGAGTGTAGTAAACA
TTCCAAAACCGTGATGGAATGGAACAACACTTAAAATCGCAGTATCCGGA
ATGATTTGATTGCCAAAAATAGGATCTCTGGCATGCGAGAATCTGACGCA
GGCAGTTCTATGCGGAAGGGCCNACCCTTAGGTAACCCAGTA

rv
TACTGGGTTACCTAAGGGTNGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGTCCAAATTGTAA
GCCGGCC
Not1
GCGGCCGCA
TCATGGTTTTTACCTCCTGAATTCGGATCCCTCGAGCGATACACACTTCTATAGTGTCACCTAAATGCGTTTAAACCTTCCTGCAGGTGACGATTACCTAACAATCGGTCGNNNNNNNANNNNNNN
Score Expect Identities Gaps Strand
1960 bits(1061) 0.0 1065/1067(99%) 1/1067(0%) Plus/Plus
Query 1 TACTGGGTTACCTAAGGGT-NGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCA 59
||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||
Sbjct 629 TACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCA 688

Query 60 TGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGT 119
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 689 TGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGT 748

Query 120 TCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCG 179
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 749 TCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCG 808

Query 180 AGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAA 239
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 809 AGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAA 868

Query 240 AATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGAT 299
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 869 AATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGAT 928

Query 300 TGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAA 359
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 929 TGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAA 988

Query 360 AGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGG 419
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 989 AGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGG 1048

Query 420 GCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGC 479
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1049 GCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGC 1108

Query 480 GGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAAC 539
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1109 GGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAAC 1168

Query 540 GCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTA 599
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1169 GCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTA 1228

Query 600 TGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGG 659
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1229 TGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGG 1288

Query 660 AGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCTTT 719
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1289 AGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCTTT 1348

Query 720 AATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAACA 779
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1349 AATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAACA 1408

Query 780 CCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCC 839
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1409 CCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCC 1468

Query 840 CGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTA 899
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1469 CGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTA 1528

Query 900 CGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGA 959
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1529 CGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGA 1588

Query 960 AGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAA 1019
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1589 AGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAA 1648

Query 1020 GGCCAAGAAGGGCGGAAAGTCCAAATTGTAAGCCGGCCGCGGCCGCA 1066
|||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1649 GGCCAAGAAGGGCGGAAAGTCCAAATTGTAAGCCGGCCGCGGCCGCA 1695

>210108-18_A05_1F85DAA019.ab1 1225, 1/9/2021
2889 in MiniT2 seq with pminiT-R primer

no good missing TCATATGTA Nde1 site

NNNNNNTNNNNNGTATTTAGAAATAAACAAATAGGGGTTCCGCCCGCGAA
ATTAATACGACTCACTATAGGGAGACGCACAT
Not1
GCGGCCGC CTCGAGAATT
CTGACGTCTTAATTAATTATTATC

AGGCGCGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCG
CTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGA
ACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGA
ATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGC
TGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTC
TTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGC
GAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGC
AGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAAC
GTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAA
AACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATC
TACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGT
GACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACC
TAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATG
CCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTA
AGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATA
TTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGC
TGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTA
CCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGA
TTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAG
AAGTCGGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACA
AGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCCAAG
GGGGAAGATAAAACCGGGCGCGGGC

Alignment statistics for match #1
Score Expect Identities Gaps Strand
1989 bits(1077) 0.0 1089/1094(99%) 3/1094(0%) Plus/Plus
Query 130 AGGCGCGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGA 189
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 20 AGGCGCGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGA 79

Query 190 GGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGG 249
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 80 GGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGG 139

Query 250 AACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGA 309
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 140 AACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGA 199

Query 310 AATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAAT 369
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 200 AATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAAT 259

Query 370 CGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTAT 429
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 260 CGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTAT 319

Query 430 CGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTAT 489
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 320 CGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTAT 379

Query 490 GAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAA 549
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 380 GAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAA 439

Query 550 CGTGCaaaaaaaaTTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTA 609
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 440 CGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTA 499

Query 610 CCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGA 669
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 500 CCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGA 559

Query 670 ATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTC 729
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 560 ATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTC 619

Query 730 CTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAG 789
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 620 CTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAG 679

Query 790 ATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTT 849
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 680 ATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTT 739

Query 850 AAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATG 909
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 740 AAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATG 799

Query 910 TGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCA 969
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 800 TGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCA 859

Query 970 GGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAG 1029
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 860 GGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAG 919

Query 1030 CACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACC 1089
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 920 CACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACC 979

Query 1090 TCTTTCGAAAGAAGTCGGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGAC 1149
|||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||
Sbjct 980 TCTTTCGAAAGAAGTC-GGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGAC 1038

Query 1150 AAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCCAAGGGGGAAGAT 1209
||||||||||||||||||||||||||||||||||||||||||||||| ||||||| |||
Sbjct 1039 AAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCG-AGGGGGATGAT 1097

Query 1210 AAAACCGGGCGCGG 1223
||| ||||||||||
Sbjct 1098 AAA-CCGGGCGCGG 1110

PCR again using

10 uM 2895-F3 TCTAGTCCGCCAAGTCTAGcatatgtaaGGCGCGCCCAC
1uL 2889-F: TCATATGTAAGGCGCGCCACCATGGAAGACGCCAAAAACA
and
10 uM 2889-R: ttGCGGCCGCGGCCGGCTTACAATTTGGACTTTCCG
(CGGAAAGTCCAAATTGTAAGCCGGCCGCGGCCGCaa)
clone into pMiniT, then subclone using Nde1-Not1

>210119-09_E07_1F85DAA000.ab1 1347, 1/20/2021
2889 in pVEE good
NNNNNNNNNNNTNNGCTACCTGATGGACTACGACATAGTCTAGTCCGCCA
AGTCTAG
Nde1
CATATG TAAGGCGC
Kozak
GCCACCATGGAAGACGCCAAAAACATAAA
GAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGC
AACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCT
TTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGA
AATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAA
ATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCG
GTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACAT
TTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCG
TAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAA
AAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTA
CCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCG
GTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACA
ATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGT
GGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATC
CTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTT
CCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATG
TGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTAC
GATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTA
TTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAA
TTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGG
AAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATATGGG
CTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAA
ACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTGGGGA
TCTGGATACCGGGAAAAANCTGGGCGTTAATCAGAAGGGGAATTAGGNTC
AAAAGACCTTGAATTAGTCCGGTTTGTAAACANTCCGGAAGNNCCACCCC
TTGATGANAGGNNGGNGGGTNNNTNNGGGAAANNNNTTTANNGGAAA

Score Expect Identities Gaps Strand
2178 bits(1179) 0.0 1221/1242(98%) 9/1242(0%) Plus/Plus
CATATGTAAGGCGCGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAG 131
|||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CATATG-AAGGCGCGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAG 98

Query 132 GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGA 191
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 99 GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGA 158

Query 192 ACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAA 251
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 159 ACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAA 218

Query 252 ATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATC 311
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 219 ATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATC 278

Query 312 GTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATC 371
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 279 GTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATC 338

Query 372 GGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATG 431
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 339 GGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATG 398

Query 432 AACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAAC 491
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 399 AACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAAC 458

Query 492 GTGCaaaaaaaaTTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTAC 551
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 459 GTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTAC 518

Query 552 CAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAA 611
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 519 CAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAA 578

Query 612 TACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCC 671
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 579 TACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCC 638

Query 672 TCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGA 731
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 639 TCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGA 698

Query 732 TTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTA 791
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 699 TTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTA 758

Query 792 AGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGT 851
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 759 AGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGT 818

Query 852 GGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAG 911
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 819 GGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAG 878

Query 912 GATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGC 971
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 879 GATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGC 938

Query 972 ACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCT 1031
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 939 ACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCT 998

Query 1032 CTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAA 1091
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 999 CTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAA 1058

Query 1092 GGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAA 1151
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1059 GGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAA 1118

Query 1152 CCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTGG-GGATCTGGATACC 1210
|||||||||||||||||||||||||||||||||||||||||||| | |||||||||||||
Sbjct 1119 CCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACC 1178

Query 1211 GGGAAAAANCTGGGCGTTAATCAGA-AGGGGAATTA-GGNTCAAAAGACCT-TGAATTA- 1266
||||||| |||||||||||||||| ||| |||||| | ||| | ||||| ||| |||
Sbjct 1179 GGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGA-TTAT 1237

Query 1267 GTCCGGTT-TGTAAACANTCCGGAAGNN-CCACC-CCTTGAT 1305
|||||||| |||||||| |||||||| ||| | |||||||
Sbjct 1238 GTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGAT 1279

Range 2: 1 to 38GraphicsNext MatchPrevious MatchFirst Match
Alignment statistics for match #2
Score Expect Identities Gaps Strand
65.8 bits(35) 4e-14 38/39(97%) 1/39(2%) Plus/Plus
Query 58 96
|||||| ||||||||||||||||||||||||||||||||
Sbjct 1 38
</pre>

2885

2022-03-26T12:55:36Z

Paul.xu:

<pre>
PCR frag 1 from the gene synthesis fragment

10 uM 2887-F3
taatctagaacgcgTCGAGGCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG
1 uM 2887-F4
GGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGAATTAATTCTTGAAGACG

2887-R1
CATCCTTATAGTGAGTCGTATTAGAG
(CTCTAATACGACTCACTATAAGGATG)
--> 1888 bp
taatctagaacgcgTCGAGGCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG AGGGGTTTTTTGGAATTAATTCTTGAAGACG AAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTCTAATACGACTCACTATAAGGATG

frag 2
2887-F2
CTCTAATACGACTCACTATAAGGATGGGCGGCGCATG

2887-R2
GAGCTGCATAGGCCTATGTTACGAGCCGTTAAC
(GTTAACGGCTCGTAACATAGGCCTATGCAGCTC)

--> 711 bp
CTCTAATACGACTCACTATAAGGATGGGCGGCGCATG
AGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTC
_______________
PCR again using the mix of two fragments
2887-F1
AAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGG
2887-R2
GAGCTGCATAGGCCTATGTTACGAGCCGTTAAC
(GTTAACGGCTCGTAACATAGGCCTATGCAGCTC)
--> 2593 bp
AAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGG
CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG
AGGGGTTTTTTGGAATTAATTCTTGAAGACG
AAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAG
~~ T7 AG~~
CTCTAATACGACTCACTATAAGG
ATGGGCGGCGCATG
AGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTC

Gibson mix cloning into HpaI+ Mlu1 digested vector
</pre>

[[File:2885 2895 2989 2906.JPG|RTENOTITLE]]

no good, no Nde1 site, oligo is bad

= == =

Repeat, subclone into 2906

[[File:2895 2885.JPG|RTENOTITLE]]

T7 promoter TAATACGACTCACTATAG

ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAGGCAGACGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATTCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTA VEE-seq-F AATCATTCAGCTACCTGAGAG

GGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGTCTAG

Nde1

CATATG

(T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC- for cleancap)

TCTTCTGGTCCCCACAGACTCAGAGAGAACCC   gccacc

  atggtgTCTaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa   Not1 GCGGCCGC AAGCTGCTTACATAGAACTCGCGGCGATTG  

 Sph1 GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA PacI ttaattaa Xba1 tctaga Mlu1 acgcgT CGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

<pre>
sequencing using 2887-seq-F:
TCGATTTTTGTGATGCTCGTC

VEE-pA-R2
sequencing T7 terminator using VEE-pA-R2
GGGGTTCCGCGCACATTTC

>210113-22_E03_1F85DAA010.ab1 1336 , 1/14/2021
2885
NNNNNNNNTTNNNTNCNGCTACCTGANGGACTACGACATAGTCTAGTCCG
CCAAGTCTAG
Nde1
CATATGTCTTCTGGTCCCCACAGACTCAGAGAGAACCC
GCCACC

ATGGTGTCTAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCC
TGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC
GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG
CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA
CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC
GACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCAT
CTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCG
AGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAG
GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCA
CAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT
TCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCAC
TACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA
CCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC
GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC
GGCATGGACGAGCTGTACAAGTAA
Not1
GCGGCCGCAAGCTGCTTACATAGAACTCGCGGCGATTG
GCATGC
CGCCTTAAAATTTTTATTTTATTTTTTCTTTT
CTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAA
AAAAAAAACGCGAAAAAAAAAAAAAAANAAAAAAAAAAAAAAAAAAAAAA
AAAAAATTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAATTTNTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAATATTTTTTTTTTTAAACNCGCCNAGGGGAATATTTTTNNGAAACAAA
AGGGNNNGGGGGCCTTTTTGGGGAAAGGGGGCGGGAANNCCCTTTTTGTT
TNTTTTTTAAAANNNCTAAAATTGTCCCCTNCGNNAAAAAAACCCCNNGA
AAAGGGTTCAAAATNNGNAAAAGGAAGATNNNNATATTCNNNTTTCCGGG
GCGCCCTNNNNCCTTTTTGGGNATTTNNCCCCCGGT

Score Expect Identities Gaps Strand
1851 bits(1002) 0.0 1037/1053(98%) 10/1053(0%) Plus/Plus
Query 61 CATATGTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGTGTCTAAGGGCG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1 CATATGTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGTGTCTAAGGGCG 60

Query 121 AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 61 AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC 120

Query 181 ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121 ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGA 180

Query 241 AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 181 AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA 240

Query 301 CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 241 CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA 300

Query 361 AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 301 AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCA 360

Query 421 ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGC 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 361 ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGC 420

Query 481 TGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 421 TGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT 480

Query 541 ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 481 ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT 540

Query 601 TCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 541 TCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA 600

Query 661 ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGT 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 601 ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGT 660

Query 721 CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGA 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 661 CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGA 720

Query 781 CCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCGGCCGCAAGCTGCT 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 721 CCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCGGCCGCAAGCTGCT 780

Query 841 TACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAatttttattttattttttctttt 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 781 TACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTT 840

Query 901 cttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaacg 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 841 CTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACG 900

Query 961 cgaaaaaaaaaaaaaaanaaaaaaaaaaaaaaaaaaaaaaaaaaaattt---tt--taaa 1015
||||||||||||||||| |||||||||||||||||||||||||||| || ||||
Sbjct 901 CGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTAATAAA 960

Query 1016 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaat----ttn-taaaaaaaa 1070
|||||||||||||||||||||||||||||||||||||||||| || |||||||||
Sbjct 961 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTAATAAAAAAAA 1020

Query 1071 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1103
|||||||||||||||||||||||||||||||||
Sbjct 1021 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1053

[[Category:Cloning]]

2794

2022-03-26T12:28:57Z

Paul.xu:

== mRNA ==

20200619 to produce linear template for Cleancap mRNA, cut vector 2794 with XbaI. But the vector was cut to two parts. which bands contains the SAM template is hard to judge. Next step, change to PacI to cut 2794.

[[File:961 2794 digestion.JPG|RTENOTITLE]]

[[File:2794 digestion mRNA.JPG|RTENOTITLE]]

[[File:2794 nde1 not1.JPG|RTENOTITLE]]

[[File:2794 nde1 not1 2.JPG|RTENOTITLE]]

<pre>

T7
TAATACGACTCACTATAG

ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA

NSP1 ~NSP4
ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAGGCAGACGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATTCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTA

sequencing primer: VEE-seq-F
AATCATTCAGCTACCTGAGAG

GGGCCCCTATAACTCTCTACGGCTAA

CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGTCTAG
Nde1
catatggaattccggccacc
TagGFP2
ATGGTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACGGCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCGACCACTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAA
Not1
GCGGCCGC

IRES
AACTAACTTAAGCTAGCAACGGTTTCCCTCTAGCGGGATCAATTCCGCCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATACC

E3L
ATGAGCAAGATCTACATCGACGAGCGGAGCAACGCCGAGATTGTGTGCGAGGCCATCAAGACCATCGGAATCGAAGGCGCCACAGCCGCTCAGCTGACCAGACAGCTGAACATGGAAAAGCGGGAAGTGAACAAGGCCCTGTACGACCTGCAGAGAAGCGCCATGGTGTACAGCAGCGACGACATCCCTCCTCGGTGGTTTATGACCACAGAGGCCGACAAGCCTGACGCCGATGCTATGGCCGACGTGATCATCGACGACGTGTCCCGCGAGAAGTCCATGAGAGAGGACCACAAGAGCTTCGACGATGTGATCCCCGCCAAGAAGATCATCGATTGGAAGGGCGCCAATCCTGTGACCGTGATCAACGAGTACTGCCAGATCACCAGAAGAGACTGGTCCTTCCGGATCGAGAGCGTGGGCCCTAGCAATAGCCCTACCTTCTACGCCTGCGTGGACATCGACGGCAGAGTGTTCGATAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAAAACAATGCCGCCAAGCTGGCCGTGGATAAGCTGCTGGGCTATGTGATCATCCGGTTCTAA

GGCCGC

IRES
AACTAACTTAAGCTAGCAACGGTTTCCCTCTAGCGGGATCAATTCCGCCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATACC

Puro
ATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGA

3'UTR, poly A
gaattGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
PacI
ttaattaa

Xba1
tctaga
Mlu1
acgcgT
CGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

digest with Xba1 or PacI as template for mRNA

>200624-21_G01_1E9BDAA008.ab1 1382, 6/25/2020
2794 verified

NNNNNNNNTCTNCGGCTACTGATGGACTACGACATAGTCTAGTCCGCCAA
GTCTAG
Nde1
CATATGG

AATTCCGGCCACCATGGTGAGCGGGGGCGAGGAGCTG
TTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACGGCGACGTGCACGG
CCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACGGCA
AGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGG
CCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTA
CCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGG
GCTACATCCAGGAGCGCACCATCCAGTTCCAGGACGACGGCAAGTACAAG
ACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGA
GCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGC
TGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCC
AACAACGGCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGG
CGGCGTGCAGCTGGCCGACCACTACCAGACCAACGTGCCCCTGGGCGACG
GCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATC
AGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTT
CAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAAGCGG
CCGCAACTANCTTAAGCTAGCAACGGTTTCCCTCTAGCGGGATCAATTNC
GCCCCCGCCCCTAACGTTAGGGTACGAAGCCANTTGGAAATGAGGCNNNN
GGTGCGTANGTCTANNTGTGATTTTCCACCAGAGATGGGCGCCTTTTGGA
CATGTGA
Not1
GCGGCCGg

NAAACNAGAGACTGTCTTCGTGACGAGACATTCGT
AGAGNNAGTTCNCCTCNCNACANNGATGCNAAGGCNNGTTGAATGANCGA
GTAGTAAAGACATACTCTGGAAACGTTCTGNNNGACAAAGCAGCGGACGA
TAGAGACACTTTAGCTAGNNNNNGAAGACNGCAGCNNGANAACGAGAGGN
GANTNCGGGNCANAGNACNNGTAGTATGANNNNNNACTNGCAAAGGGNNN
NCATCCCNAGGNCCNNCGTNNNNGTNNNNNNNGNNNGGANNAGAGNCNNN
GGTCCNNNCTCCAGAGCNANNTTCNANNNNNNNNCNNNAAAGNANNNNGA
NNNNNATAGCCANNANNNNNGANNNTTACNNNNNNNNGNNGNNNNAANNN
CNNNANGGTNNNNNNNNNNNNGANAGAAAANN

Score Expect Identities Gaps Strand
1598 bits(865) 0.0 943/986(96%) 8/986(0%) Plus/Plus
Query 19 CTG-ATGGACTACGACATAGTCTAGTCCGCCAAGTCTAGCATATGGAATTCCGGCCACCA 77
||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 2 CTGAATGGACTACGACATAGTCTAGTCCGCCAAGTCTAGCATATGGAATTCCGGCCACCA 61

Query 78 TGGTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACG 137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 62 TGGTGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACG 121

Query 138 GCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACG 197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122 GCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACG 181

Query 198 GCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCC 257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 182 GCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCC 241

Query 258 TGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGA 317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 242 TGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGA 301

Query 318 TGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGT 377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 302 TGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGT 361

Query 378 TCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGG 437
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 362 TCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGG 421

Query 438 TGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACA 497
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 422 TGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACA 481

Query 498 AGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACG 557
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 482 AGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACG 541

Query 558 GCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCG 617
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 542 GCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCG 601

Query 618 ACCACTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACT 677
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 602 ACCACTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACT 661

Query 678 ACCTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGC 737
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 662 ACCTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGC 721

Query 738 TCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAAG 797
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 722 TCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGTAAG 781

Query 798 CGGCCGCAACTANCTTAAGCTAGCAACGGTTTCCCTCTAGCGGGATCAATTNCGCCCCCG 857
|||||||||||| |||||||||||||||||||||||||||||||||||||| |||||||
Sbjct 782 CGGCCGCAACTAACTTAAGCTAGCAACGGTTTCCCTCTAGCGGGATCAATTCCGCCCCCC 841

Query 858 CCCC-TAACGTTAGGGTACGAAGCCANTTGGAAATGAGGCNNNNGGTGCGTANGTCTANN 916
|||| |||||||| | ||||||| |||||| | |||| |||||| |||||
Sbjct 842 CCCCCTAACGTTACTGGCCGAAGCCGCTTGGAA-TAAGGCCGGT-GTGCGTTTGTCTATA 899

Query 917 TGTGATTTTCCACCAGAGATGGGCGCCTTTTGGACATGTGAGCGGCCGNNAAACNAGAGA 976
||| ||||||||||| | || || ||||||| ||||||| | ||| |||| |
Sbjct 900 TGTTATTTTCCACCATAT-TGC-CGTCTTTTGGCAATGTGAGGGCCCGG-AAACCTGGCC 956

Query 977 CTGTCTTCGTGACGAGACATTCGTAG 1002
|||||||| ||||||| ||||| |||
Sbjct 957 CTGTCTTCTTGACGAG-CATTCCTAG 981
</pre>

Cre FLP mRNA

2022-03-23T13:03:28Z

Paul.xu:

<div>PCR FLPe from </div><div>2140 pCMV5-tat-CD63-Flpe</div><div>2139 pCMV5-pene-CD63-Flpe</div><div>2137 pCMV5-CD63-Flpe</div><div>2131 pCMV5-Gag-Flpe-IRES-VSVG-WPRE-oriP</div><div>2110 pFastBac-Dual-VSVG-HIVGag-FLPe-CMV-Flpe</div><div>2109 pCMV-RSV-Gag-Flpe</div><div> </div><div>using</div><div>12_13_2012D08T7-NLS-FLPe</div><div>12_13_2012D09FLPe-Xho-R</div><div>TAGTCGCTCGAGCTAGATCCGCCGGTTGATGTAG</div><div> </div><div><div>FLPo-BamH1-FCGATCAGGATCCGCCGCCACCATGGCTCCTAAG</div><div>FLPo-Xho1-FTAGTCGCTCGAGTCAGATCCGCCTGTTGATGTAG</div><div>T7-NLS-CreTAATACGACTCACTATAGGGATGGGCCCCCCCAAGAAGAAG</div><div>T7-NLS-FLPoTAATACGACTCACTATAGGGATGGCTCCTAAGAAGAAGAGG</div><div>T7-NLS-FLPeTAATACGACTCACTATAGGGATGGCCCCCAAGAAAAAGCGG</div><div>FLPe-Xho-RTAGTCGCTCGAGCTAGATCCGCCGGTTGATGTAG</div></div><div> </div><div>PCR FLPo from  </div><div> </div><div>12_13_2012D07T7-NLS-FLPo</div><div>TAATACGACTCACTATAGGG ATGGCTCCTAAGAAGAAGAGG</div><div>7_29_2013C05Flpo-Xho1-R</div><div>TGCTAGCTCGAGTCAGATCCGCCTGTTGATGTAG</div><div>2364 pFastBac-oriP-Flpo-PDGFR-WPRE</div><div>2179 pT7A3HC-6H-Tat-sc-MS2-Flpo</div><div>2173 pCMV5-CD16-Tat-Flpo-RIgE</div><div>2159 pJ912-MS2Pac-Flpo</div><div>2151 pEC-Flpo-PP7Pac</div><div>2150 pEC-Flpo-MS2Pac</div><div>2103 pCMV-RSV-Gag-Flpo</div><div>2032 pCold-Tat-Flpo</div><div> </div>[[Category:Protocols]]

File:Gel purification.JPG

2022-03-13T18:39:15Z

Paul.xu:

File:Fluc PCR2.JPG

2022-03-13T18:20:05Z

Paul.xu:

File:Fluc PCR.JPG

2022-03-13T18:19:48Z

Paul.xu:

2952

2022-03-06T23:16:35Z

Paul.xu:

PCR amp from 2889 using 2952-F + 2952 R, digest PCR product with Nde1 + Mlu1

digest 2952 with Nde1 + Mlu1, cut out 1991 small band

ligate and transform DH10g

[[File:2952 2953 cloning.JPG]][[Category:Cloning]]

[[File:2952 2953 colonies PCR.JPG]]

[[File:2952 2953 digestion.JPG]]

[[File:2952 PCR Screening.JPG]]

[[File:2952 digestion1.JPG]]

File:2952 2953 cloning.JPG

2022-03-06T22:58:00Z

Paul.xu:

File:2952 PCR Screening.JPG

2022-03-06T22:56:54Z

Paul.xu:

File:2952 2953 digestion.JPG

2022-03-06T22:56:40Z

Paul.xu:

File:2952 2953 colonies PCR.JPG

2022-03-06T22:56:25Z

Paul.xu:

File:2952 digestion1.JPG

2022-03-06T22:33:26Z

Paul.xu:

2953

2022-02-21T20:54:16Z

Paul.xu:

T7AG-EGFP

digest 2952 with Nde1-Xba1--> vector, 1867

Digest 2885 with Nde1-Xba1--insert 1069

ligate and transform 10G

http://18.206.161.119/eln/images/7/75/2953_cloning.JPG
[[Category:Cloning]]

File:2953 cloning.JPG

2022-02-21T20:51:35Z

Paul.xu:

Capped mRNA

2022-02-21T16:20:47Z

Paul.xu:

== Introduction ==

Most naturally occurring eukaryotic mRNAs and many viral RNAs have a modified nucleotide structure known as a "cap" on their 5' terminus. In vivo, the cap structure is involved in the initiation of protein synthesis and in mRNA processing and stability. Many viral RNAs are infectious only when capped, and uncapped RNAs introduced into cells via transfection or microinjection are rapidly degraded by cellular RNases. Therefore, RNA prepared for microinjection into oocytes or for transfection into eukaryotic cells should be capped. In vitro, capped mRNAs are translated more efficiently than uncapped transcripts in rabbit reticulocyte lysate or wheat germ translation systems. The trimethylated cap is involved in  RNA transport and RNA translation in nematodes.  Transcripts synthesized for use in in vitro RNA splicing experiments may also need to be capped.

The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 μg RNA per 20 μl reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed. Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability, efficient translation, nuclear transport and splicing. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or  co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications such as generating viral genomic RNA for reverse-genetic systems and crystallographic studies of cap binding proteins such as eIF4E.

The 5' cap structure improves stability of the RNA and translation efficiency, and hence is important for microinjection and transfection experiments. The co-transcriptional capping method generates 40-50 μg of ~80% capped RNA. It is important to note that using 3'-0-Me-m7G(5')ppp(5')G RNA cap analog (ARCA), which is blocked at the 3'-hydroxyl of the m7G ensures incorporation of the cap in the correct orientation. To cap larger quantities of RNA it is possible to scale up the standard reactions for both capping methods. However, using the Vaccinia Capping System would be a more feasible option cost-wise since co-transcriptional capping utilizes cap analog, which is a relatively expensive component. The Vaccinia Capping System is also a better method since it has capping efficiency of almost 100%

== NEB T7 mRNA Protocol ==

Kit: T7 High Yield RNA Synthesis Kit, Cat #: E2040S

'''Plasmid Templates'''

*Digest plasmid with an enzyme to generate blunt ends (EcoRV) or 5´-overhangs (e.g., BamH1). Select site should be at the downstream of the insert to be transcribed.
*After linearization, use spin column to purify DNA. Elute with water.

'''PCR product:'''

 100–500 ng of PCR fragments can be used in a 20 μl in vitro transcription reaction.

PCR primer design:

F: TAA TAC GAC TCA CTA TAG GG + 21 bp of the 5' end of GOI.

R: Gene specific primer.

 Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

Make 20 mM GTP solution by combining 2 μl of 20 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use. Prepare cap analog at the concentration of 40 mM. Assemble the reaction at room temperature in the following order:

{| cellspacing="0" cellpadding="1"
|-
| Nuclease-free water
| X μl
|  
|-
| 10X Reaction Buffer
| 2 μl
|  
|-
| ATP (100 mM)
| 2 μl
| 10 mM final
|-
| UTP (100 mM)
| 2 μl
| 10 mM final
|-
| CTP (100 mM)
| 2 μl
| 10 mM final
|-
| GTP (20 mM)
| 2 μl
| 2 mM final
|-
| Cap Analog (40 mM)
| 4 μl
| 8 mM final
|-
| Template DNA
| X μl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 μl
|  
|-
| '''Total reaction volume'''
| '''20 μl'''
|  
|}

 Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours. The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used. Table 1. Effect of cap analog:GTP ratios on RNA yield

{| cellspacing="0" cellpadding="1"
|-
| width="127" | '''Cap Analog: GTP Ratio'''
| width="138" | '''Concentration of Cap''' 
'''Analog: GTP (mM)'''

| width="113" | '''RNA Yield (μg) in''' 
'''2 hours'''

| width="97" | '''% Capped RNA'''
|-
| 0:1
| 0:10
| 180
| 0
|-
| 1:1
| 5:5
| 90–120
| 50
|-
| 2:1
| 6.7:3.3
| 60–90
| 67
|-
| 4:1
| 8:2
| 40–50
| 80
|-
| 8:1
| 8.9:1.1
| 20–25
| 89
|}

 To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes. Proceed with purification of synthesized RNA using a spin column

PCR Template Preparation 1. Cloning of IDT gene fragment into pMiniT2 or pJet1.2 2683 (DS1) TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA

 2. Make miniprep, send miniprep DNA out for sequencing (Genewiz, Macrogen) 3, PCR   1 ul of plasmid (50~100 ng) 1 ul of 10 uM T7-mRNA-F3 TAATACGACTCACTATAAGGAGG 1 ul 1 uM 2683-R2 2683-R2 TTACTTGTCGTCATCGTCTTTGTAGTCACCAGCTGTCCAACCTGAAG (CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAA) 1 ul 10 uM Flag-R  TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG (CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA) 21 ul H2O + 25 ul 2 X Phusion PCR mix (NEB)

PCR: 2 min at 95 oC 30 cycles of 95 oC 30 sec, 55 oC 30 sec, 72 oC 1 min 7 min at 72 oC. Final Product 802 bp TAATACGACTCACTATAAGGAGG ccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTT CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA MRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGDYKDDDDK

purify PCR fragment using Qiagen gel purification kit

Use 1 ug for IVT

= 5’AG 3’ for the CleanCap AG reagent, N-7113= =

[https://www.tebu-bio.com/blog/2018/03/13/tips-to-modify-mrna-for-in-vivo-applications-and-delivery-optimisation/ https://www.tebu-bio.com/blog/2018/03/13/tips-to-modify-mrna-for-in-vivo-applications-and-delivery-optimisation/]

CleanCap reagents are easy to use. They are simply compatible with wild-type T7 polymerase. You apply the same quantity for CleanCap and the rNTP (CTP, UTP, ATP, GTP). It could be 5mM of each depending on the usual protocol. Should use ENB's T7-FlashScribe in vitro transcription kit because of its high yield production (180µg from 1µg)!

CleanCap AG is optimized for production of mRNA that initiates with adenosine as the first base, guanosine as the second base, and guanosine as the third base, so AGG.

Unfortunately, most conventional transcripts start with 5’-GGG.

It would be a T7 promoter sequence like that 5’TAATACGACTCACTATAGGGNNNNNNNNNNNN… 3’.

So for optimal results with the CleanCap AG reagent, you would need have the three bases after the TATA box be AGG.

Thus the best T7 promoter sequence would be 5’TAATACGACTCACTATAAGGNNNNNNNNNNNN… 3’

Using that sequence with the CleanCap AG reagent, N-7113, you will get 95-98% capped mRNA with the natural CAP1 structure.

Keeping the benefit of CAP1 structure If you cannot optimize the T7 promoter sequence, you should use the CleanCap GG reagent, N-7133.

The capping efficiency would be similar to the ARCA (it could be 80%), but you will still benefit of a natural CAP1 structure in place of the CAP0 with ARCA. CAP1 boosts translation compare to CAP0 in most cell types.

#2 – Pseudo-UTP and 5-Methyl-CTP to reduce immune response of cells against mRNA

We offer a variety of modified rNTPs suitable for in vitro transcription. This includes the Pseudo-UTP and 5-Methyl-CTP used to reduce cellular response against mRNA

It is now well established that a full substitution with these two modified rNTP reduces the innate immune response of cells. Such modRNA, as called by Pr Chien’s team in their article Nat. Biotechnol. in 2013 (10.1038/nbt.2682), can be used as treatment in vivo with no more need for HPLC purification

They showed that intramyocardial injection of VEGF-A coding modRNA after myocardial infarction (MI) induces vascular regenration (see picture below).

TriLink’s teams and partners have made further investigations and found that the 5-methoxy-UTP fully substitute mRNA can also efficiently reduce the immune response of the cells and the toxicity of the modified mRNA. The use of 5-methoxy-UTP (5moU) substitution is license free.

To track mRNA in vivo and optimise their delivery into cells, Cy5 labelling is possible though translation efficiency correlated inversely with Cyanine 5-UTP substitution.

Transcription with Cyanine 5-UTP:5-Methoxy-UTP at a ratio of 1:3. Substitution in this ratio results in mRNA that is easily visualized and can still be translated in cell culture.

= =

= Capped RNA Synthesis (E2040) =

== Introduction ==

The recommended ratio of cap analog to GTP is 4:1. Cap analogs are sold separately. Please refer to the ordering information section for more information.

a 4:1 mixture of a cap analog to GTP in transcription reactions will cap 80% of the resulting mRNAs

== Protocol ==

#Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.
#Make 20 mM GTP solution by combining 2 μl of 100 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.
#Prepare cap analog at the concentration of 40 mM.
#Assemble the reaction at room temperature in the following order:

{|
|-
| Nuclease-free water
| X μl
|  
|-
| 10X Reaction Buffer
| 2 μl
|  
|-
| ATP (100 mM)
| 2 μl
| 10 mM final
|-
| UTP (100 mM)
| 2 μl
| 10 mM final
|-
| CTP (100 mM)
| 2 μl
| 10 mM final
|-
| GTP (20 mM)
| 2 μl
| 2 mM final
|-
| Cap Analog (40 mM)
| 4 μl
| 8 mM final
|-
| Template DNA
| X μl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 μl
|  
|-
| '''Total reaction volume'''
| '''20 μl'''
|  
|}

#Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours. The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used. Table 1. Effect of cap analog:GTP ratios on RNA yield

{| cellspacing="0" cellpadding="1"
|-
| '''Cap Analog: GTP Ratio'''
| '''Concentration of Cap Analog: GTP (mM)'''
| '''RNA Yield (μg) in 2 hours'''
| '''% Capped RNA'''
|-
| 0:1
| 0:10
| 180
| 0
|-
| 1:1
| 5:5
| 90–120
| 50
|-
| 2:1
| 6.7:3.3
| 60–90
| 67
|-
| 4:1
| 8:2
| 40–50
| 80
|-
| 8:1
| 8.9:1.1
| 20–25
| 89
|}

#''Optional:'' To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.
#Proceed with purification of synthesized RNA or analysis of transcription productss by gel electrop to

Capped analog: Anti-Reverse Cap Analog (ARCA)

NEB: 3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog, S1411S, $151/µmol

[https://www.trilinkbiotech.com/arca.html https://www.trilinkbiotech.com/arca.html]  ARCAN-7003, N-7003-1$142/ µmole 

[https://www.trilinkbiotech.com/products-services/nucleoside-triphosphates-nucleotides/cap-analogs.html https://www.trilinkbiotech.com/products-services/nucleoside-triphosphates-nucleotides/cap-analogs.html]

= mRNA Synthesis with Modified Nucleotides (E2060) =

Modified mRNAs containing 5mCTP and Pseudo-UTP have been shown to suppress RNA-mediated innate immune activation in vivo. The HiScribe T7 ARCA mRNA Kit (with tailing) is capable of incorporation of 5mCTP and Pseudo-UTP, into mRNA. Up to 2.5 mM total modified nucleotides can be added into the transcription reaction without impacting the mRNA yield significantly. Other modified UTP or CTP may also be used but RNA yield will vary depending on the properties of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Please note modified nucleosides are not supplied with the kit. The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP.

#Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order:

'''Composition of NTP/cap analog mixture ('''

{|
|-
| colspan="1" rowspan="1" | '''Component'''
| colspan="1" rowspan="1" | '''Stock concentration (mM)'''
| colspan="1" rowspan="1" | '''Final concentration (mM)'''
| colspan="1" rowspan="1" | '''Volume (µl)'''
|-
| colspan="1" rowspan="1" | ATP (from MEGAscript T7 Kit)
| colspan="1" rowspan="1" | 75
| colspan="1" rowspan="1" | 7.5
| colspan="1" rowspan="1" | 4
|-
| colspan="1" rowspan="1" | GTP (from MEGAscript T7 Kit)
| colspan="1" rowspan="1" | 75
| colspan="1" rowspan="1" | 1.875
| colspan="1" rowspan="1" | 1
|-
| colspan="1" rowspan="1" | Me-CTP (from Trilink)
| colspan="1" rowspan="1" | 100
| colspan="1" rowspan="1" | 7.5
| colspan="1" rowspan="1" | 3
|-
| colspan="1" rowspan="1" | Pseudo-UTP (from Trilink)
| colspan="1" rowspan="1" | 100
| colspan="1" rowspan="1" | 7.5
| colspan="1" rowspan="1" | 3
|-
| colspan="1" rowspan="1" | 3´-O-Me-m7G(5´)ppp(5´)G RNA cap structure analog
| colspan="1" rowspan="1" | 10
| colspan="1" rowspan="1" | 2.5
| colspan="1" rowspan="1" | 10
|-
| colspan="1" rowspan="1" | Total volume
| colspan="1" rowspan="1" | 23
|}

'''IVT Reaction Mix'''

{|
|-
| colspan="1" rowspan="1" | '''Component'''
| colspan="1" rowspan="1" | '''Final concentration'''
| colspan="1" rowspan="1" | '''Amount (µl)'''
|-
| colspan="1" rowspan="1" | Nuclease-free water
| colspan="1" rowspan="1" | Variable
|-
| colspan="1" rowspan="1" | <s>RNase Inhibitor</s>
| colspan="1" rowspan="1" | <s>40 U</s>
| colspan="1" rowspan="1" | <s>1</s>
|-
| colspan="1" rowspan="1" | NTP/cap analog mixture (above)
| colspan="1" rowspan="1" | 23
|-
| colspan="1" rowspan="1" | PCR product
| colspan="1" rowspan="1" | 1 µg
| colspan="1" rowspan="1" | Variable
|-
| colspan="1" rowspan="1" | 10x reaction buffer
| colspan="1" rowspan="1" | 1x
| colspan="1" rowspan="1" | 2
|-
| colspan="1" rowspan="1" | 10x T7 RNA polymerase enzyme mix
| colspan="1" rowspan="1" | 1x
| colspan="1" rowspan="1" | 2.5-4
|-
| colspan="1" rowspan="1" | Total volume
| colspan="1" rowspan="1" | 40
|}

{|
|-
| width="33%" | Nuclease-free water
| width="33%" | to 20 µl
| width="33%" |
|-
| 2X ARCA/NTP Mix
| 10 µl
| 1 mM GTP, 4 mM ARCA, 
1.25 mM CTP, 1.25 mM UTP, >1.25 mM ATP final

|-
| 5mCTP, 10 mM
| 2.5 µl
| 1.25 mM 5mCTP final
|-
| Pseudo-UTP, 10 mM
| 2.5 µl
| 1.25 mM Pseudo-UTP final
|-
| Template DNA
| X µl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 µl
|
|-
| Total reaction volume
| 20 µl
|
|}

#Follow standard mRNA synthesis protocol.

= =

= Capped RNA Synthesis (E2040) =

== Introduction ==

The recommended ratio of cap analog to GTP is 4:1. Cap analogs are sold separately. Please refer to the ordering information section for more information.

== Protocol ==

Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

Make 20 mM GTP solution by combining 2 μl of 100 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.

Prepare cap analog at the concentration of 40 mM.

Assemble the reaction at room temperature in the following order:

{|
|-
| Nuclease-free water
| X μl
|
|-
| 10X Reaction Buffer
| 2 μl
|
|-
| ATP (100 mM)
| 2 μl
| 10 mM final
|-
| UTP (100 mM)
| 2 μl
| 10 mM final
|-
| CTP (100 mM)
| 2 μl
| 10 mM final
|-
| GTP (20 mM)
| 2 μl
| 2 mM final
|-
| Cap Analog (40 mM)
| 4 μl
| 8 mM final
|-
| Template DNA
| X μl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 μl
|
|-
| '''Total reaction volume'''
| '''20 μl'''
|
|}

Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours. The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used. Table 1. Effect of cap analog:GTP ratios on RNA yield

{|
|-
| width="25%" | '''Cap Analog: GTP Ratio'''
| width="25%" | '''Concentration of Cap''' 
Analog: GTP (mM)

| width="25%" | '''RNA Yield (μg) in''' 
2 hours

| width="25%" | '''% Capped RNA'''
|-
| 0:1
| 0:10
| 180
| 0
|-
| 1:1
| 5:5
| 90–120
| 50
|-
| 2:1
| 6.7:3.3
| 60–90
| 67
|-
| 4:1
| 8:2
| 40–50
| 80
|-
| 8:1
| 8.9:1.1
| 20–25
| 89
|}

''Optional:'' To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.

Proceed with purification of synthesized RNA or analysis of transcription productss by gel electrop

'''1. Synthesize RNA '''using MEGAscript T7 kit (Ambion, Austin, TX)

1.1 Thaw the components from the T7 High Yield RNA Synthesis kit and keep on ice.

Component Volume (μl) Final

Nuclease free water* '''Xμl'''

10X Reaction Buffer '''2μl'''

1X ATP (100 mM) '''2μl '''10mM

CTP (100 mM) '''2μl''' 10mM

UTP (100 mM) '''2μl'''  10mM

GTP (20 mM) '''2μl'''  2mM

3 ́-0-Me-m7G(5')ppp(5')G cap analog (ARCA)(40mM) '''4μl''' 8mM

Template DNA* '''Xμl''' 1μg

T7 RNA Polymerase Mix '''2μl'''

Total '''20 μl'''

'''Optionally:''' 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies, San Diego, CA) are used to replace CTP and UTP. Another protocol suggested to use 1.6 μg of purified tail PCR product as template in 40 μl reaction, and the final nucleotide reaction concentrations are 6 mM for the cap analog, 1.5 mM for guanosine triphosphate, and 7.5 mM for the other nucleotides.

1.2 Mix well by vortexing and incubate the reactions at 37 °C for 2 hours in a dry air incubator.

 '''2. Removal of Template'''

DNA The transcription reactions are treated with DNase I (RNase-free) to remove the DNA template before proceeding with purification.

2.1. Add 70 μl nuclease free water to the transcription reactions followed by 10 μl of DNase I reaction buffer.

2.2. Add 2 μl DNase I to the reactions.

2.3. Incubate at 37 °C for 15 minutes in a dry air incubator.

 '''3. Column Purification of Capped RNA'''

3.1. Purify the capped (or uncapped) RNA using MEGAclearKit or Qiagen RNeasy Mini Kit.

3.2. Quantify the RNA using a NanoDrop Spectrophotometer.

3.3. As per  manufacturer's instructions, assess RNA sample quality

(Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer was suggested but we do not have these).

'''4. RNA Capping using  Vaccinia Capping System'''

4.1. Use 10 μg of the purified capped mRNA. A standard capping reaction can cap up to 10 μg (100nt or larger) RNA. If a larger amount of RNA needs to be capped the reaction can be scaled up accordingly.

4.2. Increase the volume to 15 μl using nuclease free water.

4.3. Heat RNA solution at 65 °C for 10 minutes to remove secondary structures. Place on ice for 5 minutes.

4.4. To set up capping reaction, add 2 μl 10X Capping Buffer, 1 μl GTP (10 mM), 1 μl freshly diluted S-adenosyl methionine (2 mM) and 1 μl (10 units) Vaccinia capping enzyme to the 15 μl denatured RNA.

4.5. Mix well by gently vortexing and incubate  at 37 °C for 30 minutes in a dry air incubator.

4.6. Add 1.2 μl of 2 mM ZnCl2 (20 X) to final conc of 0.1 mM,  or 2.3 μl 10X Antarctic Phosphatase Buffer, then add 1 μl of Antarctic Phosphatase and  incubate at 37 oC for 30 min.

 '''5. Column Purification of Capped RNA'''

Purify capped RNA using MEGAclear Kit or Qiagen RNeasy Mini Kit

'''6. Quantitate RNA using Nanodrop and adjust to 100 ng/μL working concentration by addition of Tris-EDTA (TE, pH 7.0)'''

'''7. Assess RNA sample quality''' (such as Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer).

'''8. Store RNA at -80 oC.'''

2013-3-1

Lane 1: PCR   Lane 2:plasmid

[[File:RNA 3-1-2013.JPG|RTENOTITLE]]

Lane 1,2:PCR  Lane3:plasmid [[File:Capped mRNA 3-1-2013.JPG|RTENOTITLE]]

== DNA template Preparation ==

Plasmid with T7 promoter: linearize DNA using a restriction enzyme. Make sure the enzyme does not cut insert. Purify and measure DNA concentration.

Plasmid without T7 promoter: PCR insert using

'''F primer:'''

5' TAATACGACTCACTATAGGG--21 nt of gene specific sequence 3'

'''R primer:''' 5'- gene specific sequence--3'

== Synthesis of Capped Cre Recombinase mRNA ==

=== ===

This protocol describes the synthesis of Cre recombinase mRNA from a DNA template containing the Cre gene. Briefly, generic 5′ and 3′ UTRs are ligated to the Cre open reading frame (ORF). Ligation is facilitated by the use of splint oligonucleotides that bridge the junctions between the UTRs and the ORF. To this newly synthesized product, a tract of 120 Ts is appended by PCR. The final construct is amplified and used to template in vitro transcription of mRNA.

=== Materials ===

Cre ORF PCR

pBABE-puro-Cre (kindly provided by Dr. Zhe Li, Harvard Medical School)

KAPA HiFi Hotstart ReadyMix (Harvard Biopolymers – Catalog #KK2601)

5′ ORF Oligo - 5′TCCAATTTACTGACCGTACACC3′

3′ ORF Oligo - 5′CTAATCGCCATCTTCCAGCAGG3′

ORF Forward Primer Kinase Treatment

T4 Polynucleotide Kinase (PNK) with Buffer (New England Biolabs – Catalog #M0201)

Adenosine-5′-Triphosphate (ATP) (100 mM, USB (Affymetrix) – Catalog #77241)

5′ UTR and 3′ UTR Ligation

Cre Amplicon

5′ UTR Oligo

- 5′TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATG3′

3′ UTR Oligo, Phosphorylated

-5′TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATG3′

5′ Splint Oligos

- 5′GGTGTACGGTCAGTAAATTGGACATGGTGGCTCTTATATTTCTT3′

3′ Splint Oligos

- 5′CCCGCAGAAGGCAGCGATTAGCGGTAGAAGGTCGT3′

Ampligase Enzyme with Buffer (Epicentre Biotechnologies – Cat. No. A32750)

PCR to add poly-T tail (Tail PCR)

KAPA HiFi Hotstart Readymix (Harvard Biopolymers – Cat. No. KK2601)

Tail Forward Primer (Generic)

- 5′TTGGACCCTCGTACAGAAGCTAATACG3′

Tail Reverse Primer (Generic) PAGE Purified

- 5′T120CTTCCTACTCAGGCTTTATTCAAAGACCA3′

In Vitro Transcription

T7 MEGAscript Kit (Ambion – Catalog #AM1334M)

Adenosine-5′-Triphosphate (ATP) (100 mM, USB (Affymetrix) – Catalog #77241, 75 mM, included in T7 MegaScript Kit)

Guanosine-5′-Triphosphate (GRP) (100 mM, USB (Affymetrix) – Catalog #77243, 75 mM, included in T7 MegaScript Kit)

Pseudouridine-5′-Triphosphate (100 mM, TriLink Biotechnologies – Catalog #N-1019)

5-Methylcytidine-5′-Triphosphate (100 mM, TriLink Biotechnologies – Catalog #N-1014)

3′-O-Me-m7G(5′)ppp(5′)G Cap Analog (New England Biolabs – Catalog #S1411)

Tail PCR Products (100 ng/μL)

RNA Purification

MEGAclear Kit (Ambion – Catalog #AM1908M)

Phosphatase Treatment

Antarctic phosphatase (New England Biolabs – Catalog #M0289)

=== Cre Recombinase ORF PCR Amplification ===

1. Perform 5′ phosphorylation of the Cre ORF Forward primer. Combine the following: 300 pmol primer (3 μL of 100 μL stock) 1 μL (10U) PNK enzyme 5 μL 10x buffer 50 nmol ATP (0.5 μL of 100 μL stock) Incubate at 37 °C for 30 minutes Heat-inactivate at 65 °C for 20 minutes Add 250 μL TE to make 1 μM stock (100 μL to make 2 μM stock)

 2. Amplify the Cre Recombinase ORF by PCR amplification. Perform a Gradient PCR around the Tm of the primers. Perform 25 μL reactions by combining the following: 12.5 μL 2x KAPA HiFi Hotstart ReadyMix 3.75 μL Phosphorylated ORF Forward Primer (2 μM) 3.75 μL ORF Reverse Primer (2 μM) Template DNA – 10–20 ng H2O to bring final volume to 25 μl Thermal cycler profile: 95 °C 5 min 98 °C 20 sec An external file that holds a picture, illustration, etc. Object name is nihms378773t1.jpg Object name is nihms378773t1.jpg 25 cycles Gradient 15 sec 72 °C 60 sec 72 °C 5 min 4 °C Hold Analyze on agarose gel (can use 1 μL of PCR reaction diluted 10x)

 3. Perform a QIAQuick purification to purify the gel-excised DNA product (CRE recombinase). Use QIAQuick Spin Columns. Combine 5 volumes of PB Buffer to 1 volume of reaction (5:1). Mix with pipettor and apply to column. Centrifuge at 6,000 rpm for 1 minute, and discard the flow-through. Wash with 750 μL PE. Centrifuge at 6,000 rpm for 1 minute, and discard the flow-through. Centrifuge again at 6,000 rpm for 1 minute and place filter on a clean Eppendorf tube. Elute with water (nuclease-free) or EB buffer. Wait ≥ 1 minute before centrifuging at 6,000 rpm for 1 minute.

 4. Quantify and quality control PCR product using Nanodrop machine.

=== UTR Ligation ===

5. Ligate the UTRs to the CRE recombinase ORF amplicon isolated above. Begin by combining the following for a 20 μL reaction: 4 μL 5′ UTR (1 μM) 4 μL 3′ UTR-Phosphorylated (1 μM) 2 μL 5′ Splint (1 μM) 2 μL 3′ Splint (1 μM) 2 μL 10x Buffer 1 μL Ampligase 1 μL CRE ORF amplicon 4 μl Water 6. Thermal profile for UTR Ligation: 95 °C 10 sec An external file that holds a picture, illustration, etc. Object name is nihms378773t2.jpg Object name is nihms378773t2.jpg 5 cycles 45 °C 60 sec 50 °C 60 sec 55 °C 60 sec 60 °C 60 sec 4 °C Hold

Perform QIAQuick Purification as described above in the Cre Recombinase ORF PCR amplification protocol.

NOTE: It may be desirable to TA clone the newly generated UTR-flanked Cre amplicon into a plasmid to avoid the need to re-amplify from the Cre DNA plasmid. This can be accomplished by using the pcDNA3.3-TOPO TA Cloning Kit.

NOTE: Check and ensure the ligation worked by diagnostic PCR by using flanking primers and subsequently performing gel electrophoresis to double-check if the product is of the expected size. This is a critical juncture for troubleshooting—if the ligation is unsuccessful (it often requires multiple attempts) try either a wider/smaller gradient of temperatures or try a sequential ligation. The difficulty lies in that the two splints usually have different annealing conditions so both will not stick at once.

=== Tail PCR ===

7. This section will append 120 Ts to the above synthesized Cre ORF with newly ligated 5′UTR and 3′UTR. Begin by diluting the PCR product (Cre ORF with 5′ UTR and 3′UTR) to 10 ng/μL.

 8. Perform a 25 μL PCR reaction by combining the following: 12.5 μL 2x KAPA HiFi Hotstart ReadyMix 1 μL Tail Forward Primer (5 μM) – final concentration is 200 nM 1 μL Tail Reverse T120 Primer (5 μM) – final concentration is 200 nM PCR product (10 ng/μL) – 10–20 ng H2O to bring final volume to 25 μl

 9. Thermal cycler profile for Tail PCR reaction: 95 °C 5 min 98 °C 20 sec An external file that holds a picture, illustration, etc. Object name is nihms378773t3.jpg Object name is nihms378773t3.jpg 25x 65 °C 15 sec 72 °C 45 sec 72 °C 10 min 4 °C Hold

 10. Analyze the PCR product via gel electrophoresis (can use 1 μL of PCR reaction diluted 10x).

 11. Perform QIAQuick Purification as described above in the Cre Recombinase ORF PCR amplification protocol.

 12. Quantify and quality control the purification product by Nanodrop and dilute to 100 ng/μL

=== In Vitro Transcription of Modified Cre RNA ===

13. This section is for the in vitro transcription of modified Cre mRNA using the above stated protocol. Begin by preparing a nucleotide triphosphate (NTP) mix. This mix recipe is sufficient for 5.4 reactions and this proportion of Cap:GTP allows for ~80% of transcripts to be capped, though this can contribute to reduced yield. The cap powder should be dissolved in 35 μL H2O. The recipe is as follows: Mix Concentration Desired Final Concentration Cap 15 mM 6 mM GTP 3.75 mM 1.5 mM ATP 18.75 mM 7.5 mM 5methylCTP 18.75 mM 7.5 mM PseudoUTP 18.75 mM 7.5 mM

14. For a 2x reaction (40 μL), combine reagents in the following order in a PCR strip at room temperature. The NTP Mix and template should be thawed on ice, then moved to room temperature (but only for setting up the IVT, immediately return to ice afterward). The 10x Buffer can be pre-warmed to 37 °C. It is important to ensure there is no precipitate in the 10x Buffer. The template concentration should be pre-adjusted at 100 ng/μl. Custom NTP Mix 16 μL 10x Buffer (Kit) 4 μL Template (Tail PCR) 16 μL (1.6 μg) T7 Enzyme Mix (Kit) 4 μL Total 40 μL

15. Incubate the reaction at 37 °C for up to 12 hours.

16. Perform DNase treatment with 2 μL TURBO DNase (Kit) for each reaction. Gently mix and incubate at 37 °C for 15 min.

17. Perform purification with MEGAclear Kit as described in the manufacturer’s instructions: Begin by pre-warming the 115 μL Elution Solution per reaction at 95 °C using PCR machine.

18. Bring the in vitro transcription (IVT) reaction volume to 100 μL by adding 58 μL of Elution Solution. Mix by pipetting and transfer the total volume to an RNase-free Eppendorf tube.

19. To the new RNase-free Eppendorf tube, add 350 μL Binding Solution Concentrate and mix by pipetting.

20. Add another 250 μL of 100% ethanol solution and mix by pipetting.

21. Transfer the sample to a filter cartridge (assembled on a collection tube).

22. Centrifuge for 1 min at 10,000 RPM. Take care to avoid the escape of glass fibers out of the column and into the collection tube. Discard the flow-through.

23. Wash each reaction with 500 μL Wash Solution and centrifuge for 1 min at 10,000 RPM. Discard the flow-through.

24. Repeat wash with 500 μL Wash Solution. Discard the flow-through.

25. Dry by centrifuging for 30 seconds at 10,000 RPM.

26. Place filter on a new collection tube and elute RNA by adding 50 μL of 95 °C Elution Buffer. Centrifuge for 1 min at 10,000 RPM.

27. Repeat the elution step with 50 μL of 95 °C Elution Buffer. Centrifuge for 1 minute at 10,000 RPM.

28. Quantify the RNA using a Nanodrop machine before proceeding to Antarctic phosphatase treatment and a second of purification by the MEGAclear Kit.

29. Perform Antarctic Phosphatase treatment by adding 10 μL 10x buffer to each reaction. Then add 3 μL Antarctic Phosphatase. Gently mix and incubate the reaction for 30 minutes at 37 °C. Optional: Inactivate reaction at for 5 minutes at 65 °C.

31. Perform RNA Purification with MEGAclear Kit as described above (Step 17 – 27).

32. Quantify the RNA using Nanodrop. Each 2x reaction will generally produce ~50 μg RNA (the expected yield of a 40 μL reaction is generally 40 – 60 μg).

33. Adjust the RNA concentration to 100 ng/μL using RNase-free TE buffer.

34. Aliquot RNA and store at −20 °C or −80 °C. −20 °C is preferable to reduce freeze/thaw cycles.

Note: To quality control RNA, load the RNA and run gel electrophoresis under native conditions. High quality RNA should result in the observation of a single band.

=== ===

=== Cre RNA Transfection ===

1.  After allowing one or two days for recovery of pE4-hiPSCs from plating and single cell-dissociation, prepare to transfect with mRNA continuously for seven days.

 2.  Dilute 100 ng/μl of Cre RNA 5x in Opti-MEM basal media. Dilute 5 μl of RNAiMAX per microgram of RNA 10x in Opti-MEM. Combine these two dilutions and incubate at room temperature for 15 minutes.

 3.  Take the six-well plate(s) of pE4 hiPSCs out of the 37 °C incubator. Feed each well with 2 ml of fresh mTeSR1 media and 50 – 100 ng/ml of B18R interferon inhibitor.

 4.  After changing the media, add RNA/RNAiMAX complex directly to the media.

 5.  After 4 – 6 hours, refresh each well with 2 ml of mTeSR1 media and 50 – 100 ng/ml of B18R interferon inhibitor. All media changes should contain B18R when RNA transfection is occurring.

 6.   Incubate the plates in the incubator overnight. and repeat this procedure (Step 11 – 14) every day for 7 days.

 7.   After the seventh day of RNA transfection, allow putative Cre-excised hiPSC clones to expand into colonies. After observing colony formation, seed a 6-well gelatinized plate with CF1 MEF feeder cells.

== Radioactive capped mRNA ==

5′ Cap analog (10 mM) In addition to “conventional” cap analogs, which can be incorporated in two ways to produce mixtures of differently capped mRNAs, Ambion sells an antireverse cap analog (ARCA), which ensures that the cap is in the right orientation. [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) or [α-32P]UTP (800 Ci/mmol; 10 μCi/μL) We recommend labeling capped transcripts with [α-32P]GTP because the concentration of GTP is already lowered for capping to be more efficient. ATP, GTP, UTP, and CTP stocks (10 mM each, pH 7.0) It is possible to initiate transcription with dinucleotides, i.e., UpG or CpG, or even mononucleosides (i.e., G). Transcripts initiated in this way can be 5′ -end labeled without prior phosphatase treatment. Dithiothreitol (DTT) (1 M) DNase I (10 units/μL) EDTA (0.5 M) Ethanol (100%) GlycoBlue (15 μg/μL) Phenol:chloroform:isoamyl alcohol (PCA; 25:24:1) RNasin (RNase inhibitor; 40 units/μL) SDS extraction buffer <R> Sodium acetate (3 M, pH 5.2) T3 or T7 RNA polymerase (depends on template promoter; 50 units/μL) Template DNA (linearized plasmid or gel-purified PCR product) Transcription buffer for RNA polymerase (5×) <R> Equipment Dry ice Gel-purification equipment Incubator preset to 37 ̊C Micro Bio-Spin Columns with Bio-Gel P-6 in Tris buffer (Bio-Rad) Microcentrifuge Microcentrifuge tubes (1.5 mL, polypropylene, capped)

 H2O to 50 μL

Template DNA (linearized plasmid or PCR product) 2 μg

0.04 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (10 mM) 2.5 μL

0.5 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/μL) 1 μL

0.8 unit/μL DTT (1 M) 1 μL

20 mM T3 or T7 RNA polymerase (50 units/μL) 1 μL 1 unit/μL 

To monitor the nonradioactive transcription reaction, remove 10 μL of the reaction and transfer to a separate tube. Add 1 μL of [α- 32P]GTP or UTP (800 Ci/mmol; 10 μCi/μL) and process in parallel with unlabeled GTP. For trace labeled, add 0.2–0.5 μCi of GTP or UTP (800 Ci/mmol). Remove and set aside 1 μL from any labeled reaction to use for input quantitation. This reaction should yield  10–20 μg of RNA at 100 cpm/ng

For high-specific-activity RNA, combine the following in the order listed:  H2O to 50 μL Template DNA (linearized plasmid or PCR product)

2 μg 0.04 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (0.5 mM) 2.5 μL

25 μM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/μL) 1 μL

0.8 unit/μL DTT (1 M) 1 μL

20 mM [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 10 μL

2 μCi/μL T3 or T7 RNA polymerase (depends on template promoter) (50 units/μL) 1 μL 1 unit/μL 

If labeling with [α- 32P]UTP, use 2.5 μL of 10 mM GTP (instead of 0.5 mM) and 2.5 μL of 0.5 mM UTP (instead of 10 mM) and add 10 μL of [α- 32P]UTP in the above reaction instead of [ 32P]GTP. Remove and set aside 1 μL to use for input quantitation. This reaction will yield 3 μg of RNA at 50,000–100,000 cpm/ng.

For moderate-specific-activity RNA, combine the following in the order listed:

 H2O to 50 μL

Template DNA (linearized plasmid or PCR product)

2–4 μg 0/04–0.08 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (0.5 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/μL) 1 μL 0.8 unit/μL DTT (1 M)

1 μL 20 mM [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 1–4 μL

1 unit/μL T3 or T7 RNA polymerase (depends on template promoter) (50 units/μL) 5 μL 1 μCi/μL

Adjust the amount of [α- P]GTP to get the desired specific activity. This reaction will yield RNA at 4000–8000 cpm/ng.

2. Incubate the reaction for 1–2 h at 37 ̊C. 3. Add 1 μL of 10 units/μL (for each 50 μL transcription reaction) DNase I and incubate for 15 min at 37 ̊C. 4. Add EDTA to 10 mM. 5. For labeled reactions, remove unincorporated nucleotides with a Micro Bio-Spin Column and then dilute to 200 μL with SDS extraction buffer. For unlabeled or trace-labeled reactions, dilute with an equal volume of SDS extraction buffer. Add 1/10 volume of 3 M sodium acetate (pH 5.2) to both labeled and unlabled reactions. 6. Extract with PCA (see Purification of RNA by SDS Solubilization and Phenol Extraction [Rio et al. 2010]). 7. Add 2 μL of 15 μg/μL GlycoBlue or glycogen to serve as carrier or to aid in resuspending the pellet. 8. Add 2.5 volumes of ethanol. Freeze on dry ice or in dry ice/ethanol bath ( 15 min). Thaw. Centrifuge at maximum speed in a microcentrifuge for 10 min. Carefully pipette off the supernatant and dispose off by an appropriate waste method.

9. Resuspend the pellets in 2 mM EDTA (2–5 μL for each 50 μL of transcription reaction for gel purification). If RNA is to be used directly, resuspend in H2O.

10. Gel-purify the RNA.

Check a small aliquot of longer transcripts (>600–700 nucleotides) on the gel for intactness. If the RNA is a single band with no trailing of radioactivity below, the transcripts can be used without gel purification.

Capping of In Vitro-Synthesized Transcripts For capping, mix the following reagents in the order listed:

Template DNA (linearized plasmid or PCR product) 2–4 μg

0.04–0.08 μg/μL ATP (10 mM) 2.5 μL

0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 2.5 μL

0.5 mM GTP (1 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/mL) 1 μL

0.8 unit/μL DTT (1 M)

1 μL 20 mM Cap analog (see above) (40 mM)

1.25 μL 1 mM T3 or T7 polymerase (50 units/μL)

1 μL 1 unit/μL [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 5 μL

1 μCi/μL H2O to 50 μL

Substitute any cap analog, dinucleotide (NpG), or GMP for 7mGpppG at a final concentration of 1 mM. 2. Perform Steps 2–10 above.

Random Incorporation of Modified Nucleotides during In Vitro Transcription The following is a “typical” in vitro transcription reaction designed to obtain a capped, moderate-specific-activity, biotin- or 4-thio-U-substituted RNA; the modified uridine is incorporated at approximately every fourth uridine residue. See Discussion.

1. For substitution, mix the following reagents in the order listed:

Template DNA (linearized plasmid or PCR product) 2–4 μg

0.04–0.08 μg/μL ATP (10 mM) 2.5 μL 0.5 mM CTP (10 mM) 2.5 μL

0.5 mM UTP (10 mM) 3.75 μL

0.375 mM Biotin-UTP or 4-thio-UTP (1 mM)

6.25 μL 0.125 mM GTP (1 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL

1× RNasin (40 units/mL) 1 μL

0.8 unit/μL DTT (1 M) 1 μL

20 mM Cap analog (see above) (40 mM) 1.25 μL

1 mM T3 or T7 polymerase (50 units/μL) 1 μL

1 unit/μL [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 5 μL

1 μCi/μL H2O to 50 μL

2. Perform Steps 2–10 above.

Problem (Step 9): The RNA does not completely resuspend. Solution: Resuspend the RNA in a larger volume or dilute it and reprecipitate with ethanol. Very efficient transcriptions may produce a PPi precipitate in large amounts and interfere with solubility. This problem can be overcome by addition of pyrophosphatase.

Problem (Step 10): The RNA yield is low. Solution: Rerun the reaction, making sure that the mixture remains in solution while assembling the reaction. The reactions are assembled at room temperature in Step 1 to avoid precipitation. If the reactions containing high concentrations of cap and nucleotides form a precipitate, heat them for 5 min at 37 ̊C and briefly vortex before adding the enzyme.

Problem (Step 10): The RNA is degraded. Solution: Make sure that the template was nuclease-free. If necessary, after plasmid linearization, dilute 100 μL of digestion with 200 μL of SDS extraction buffer. Add proteinase K (100 μg/mL), digest for 15 min at 37 ̊C, add 1/10 volume 3 M sodium acetate (pH 5.2) and proteinase K, phenol-extract, and ethanol-precipitate. Make sure that all solutions are RNase-free.

These methods are designed to prepare RNAs by in vitro transcription. Before setting up an in vitro transcription reaction, it is necessary to have a clear idea of how much RNA is needed and at what specific activity. In addition, is the RNA to be capped or substituted? Capping is usually performed in conjunction with radiolabeling and/or with other internal modifications. The presence of a cap at the 5′ end of in vitro-synthesized transcripts can be useful in many situations, e.g., by preventing 5′ to 3′ nuclease activity or by promoting translation. “Caps” come in several forms: 7mGpppG, ApppG, and GpppG. 7mGpppG serves as a recognition factor for the translation initiation factor eIF-4E and thus is important when making RNAs that will be used for translation. The other analogs are primarily used to prevent nucleolytic attack. Capping during transcription involves the initiation of RNA chains with the cap (obviously, cap cannot be incorporated internally). The extent of capping is determined by competition between cap and GTP;

therefore, cap must be present in excess of GTP. We recommend that the concentration of cap be 10-fold that of GTP; under such conditions, 90% of the RNAs will be capped. Because of constraints on the concentrations of cap that can be achieved (1 mM is practical), it is necessary to reduce the concentration of GTP to 100 μM to achieve efficient capping.

Many modified nucleotides can be incorporated into in vitro-synthesized RNA, including, but not limited to, biotinylated UTP, 4-thio-UTP, bromo-UTP, iodo-UTP, and many adenosine analogs (with α-thiophosphates). Biotinylated RNAs are used in affinity selection experiments; 4-thio-, 5-bromo-, and 5-iodo-U-substituted RNAs are useful for cross-linking studies; and α-thio-based analogs are primarily used for modification–interference experiments. When designing in vitro transcription reactions that incorporate modified nucleotides, it is important to consider the level of incorporation desired. For example, in modification–interference approaches, transcripts should contain one or even fewer modifications per transcript, whereas in biotin applications, heavier substitution (several per transcript) is desirable. The degree of substitution is determined by competition between the unmodified and modified nucleotide just as in capping. Because some modified nucleotides are incorporated less efficiently than their unmodified counterparts, the degree of substitution may not correspond exactly to the ratio of modified to unmodified nucleotides present in the transcription reaction. Thus, it is sometimes necessary to titrate the level of the modified nucleotide to achieve the desired level of substitution. Oversubstitution with biotin can sometimes cause RNA to partition with the organic phase because of the hydrophobic spacer. Transcripts heavily substituted with biotinylated nucleotides should not be extracted with phenol.

SDS Extraction Buffer

Reagent Quantity (for 500 mL)

Final concentration Tris–HCl (1 M, pH 7.5)

10 mL 20 mM EDTA (0.5 M, pH 8.0)

1 mL 1 mM SDS (10% w/v)

25 mL 0.5% (w/v) H2O 464 mL

Transcription Buffer for RNA Polymerase (5×)

Reagent Quantity (for 10 mL) Final concentration Tris–HCl (1 M, pH 8.0) 2 mL 200 mM MgCl2 (1 M) 400 μL 40 mM NaCl (5 M) 0.5 mL 250 mM Spermidine (100 mM) 1 mL 10 mM H2O 6.1 mL 

== mRNA Precipitation ==

After adding 1U of RQ1 RNase-free DNase to the reaction and incubating at 37°C for 15 minutes, the RNA was precipitated by adding 30µl of nuclease-free water and 25µl of LiCl solution (7.5M lithium chloride, 50µM EDTA). The reaction was chilled for at least 30 minutes at –20°C and centrifuged (maximum speed in a microcentrifuge at 4°C for 15 minutes). The resulting RNA pellet was washed with 70% ethanol, dried and resuspended in 1mM sodium citrate (pH 6.4)

== mRNA transfection ==

Cells were plated in 24 well tissue culture plates (Hela 50,000 cells per well; HepG2 100,000 cells per well) in MEM with 10% FBS prior to day of transfection. The following day cells were transfected with 125 ng of mRNA with 4 ul of Messenger Max (ThermoFisher Scientific, Waltham, MA) per well following the manufacture's protocol. Appropriate volume of mRNA was dissolved in OptiMEM to make up to 100 ul. All cells were harvested at 24 or 72 hrs post transfection and processed for protein analysis.
[[Category:Protocols]]

2942

2022-02-20T22:46:51Z

Paul.xu: Created page with "need to check to see if there is a clone"

need to check to see if there is a clone

2492

2022-02-20T22:45:57Z

Paul.xu: Created page with "to be cloned or check for completed clone"

to be cloned or check for completed clone

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2022-02-20T19:49:00Z

Paul.xu: Created page with "  T7AG TAATACGACTCACTATAAGG 5'UTR tcttctggtccccacagactcagagagaacc Nde1 (cloning site 1) CATATG TAAGGCGC 5'UTR GGGAAATAAGAGAGAAAAGAAGAGTAA..."

T7AG TAATACGACTCACTATAAGG 5'UTR tcttctggtccccacagactcagagagaacc

Nde1 (cloning site 1) CATATG TAAGGCGC 5'UTR GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGG  Kozak NcoI GCCACCATGG

Stop TGATAATAG GCCGGCcgg  3'UTR AES-mtRNR1    Pan troglodytes amino-terminal enhancer of split (AES) 3'UTR    CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCG GGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC     human mitochondrial encoded 12S ribosomal RNA 3'UTR    CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAAC CTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC 

 Not1 (cloning site 2) GCGGCCGC polyA CCGCGGCCGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaattaatctagaacgcgTCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGAGCTC

 Modified 5’-cap0 structure using ARCA (Anti Reverse Cap Analog) or 5'-cap1 structure using CleanCap 5´-untranslated region  3’-UTR: comprises two sequence elements derived from the amino-terminal enhancer of split (AES) mRNA and the mitochondrial encoded 12S ribosomal RNA to confer RNA stability and high total protein expression.

N1-methyl pseudouridine is (1mt).

     (1) an ionizable or cationic lipid [e.g., SM-102 (mRNA-1273) and ALC-0315 (BNT162b2)], bearing tertiary or quaternary amines to encapsulate the polyanionic mRNA; (2) a helper lipid [LNPs of Moderna and BioNTech contain the same helper lipid 1,2-distearoyl-snglycero-3-phosphocholine (DSPC)] that resembles the lipids in the cell membrane; (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) a polyethylene glycol (PEG)-lipid [(2-[(polyethylene glycol)− 2000]-N,N-ditetradecylacetamide (PEG2000-DMA) in BNT162b2 or 1,2-dimyristoyl-rac-glycero3-methoxypolyethylene glycol-2000 (PEG2000-DMG) in mRNA-1273] to lend the nanoparticle a hydrating layer, improve colloidal stability, and reduce protein absorption.

      anti-reverse-Cap-analog [CAP-0 (N7MeGpppN) structure] or Clean Cap [CAP-1 (N7MeGpppN2′-OMe) 

 vaccinia virus immune evasion proteins E3, K3, and B18R (EKB).14,  15,  16 The latter set of genes counteracts type I interferon-dependent cellular defense mechanisms activated by mRNA delivery, which inhibit mRNA translation and may compromise cell viability upon repetitive transfection.17,  18 Transfecting these two classes of factors along with double-stranded reprogramming-enhancing microRNAs (miRNAs) from the 302/367 cluster19,  20 on 4 consecutive days has been shown to improve the efficiency and speed of generating stable iPSC lines from human neonatal and adult fibroblasts
[[Category:Cloning]]

His-Tat-NLS-Cre-Purification

2022-02-16T00:26:26Z

Paul.xu:

== Current Working Protocol ==

'''Lysis Buffer''':  25 mM   KPO4 , pH 7.5, 500 mM NaCl, 0.1% NP-40, 10 mM imidazole, 10% glycerol,  1 mM beta-ME.

'''Buffer A''',   25 mM KPO4 , pH 7.5,  500 mM NaCl, '''10 mM '''imidazole, 10% glycerol.  Store at 4 oC  .

'''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC . '''Ni Elution buffer''':  50 mM K Phosphate Buffer, '''pH 7.5''', '''200 mM NaCl''',    '''150 mM imidazole''',  10% glycerol. Store at 4 oC or in cold room.

'''Ni Elution buffer II''':  50 mM K Phosphate Buffer, '''pH 7.5''', '''100 mM Arginine or Histidine''',    '''150 mM imidazole''',  10% glycerol. Store at 4 oC or in cold room.---Need to test

'''Cre Dilution Buffer:''' 50 mM K Phosphate Buffer,  pH 7.5,  '''100 mM arginine, '''10% glycerol.  Store at 4 oC.

'''SP Washing Buffer:''' 50 mM K Phosphate Buffer,  pH 7.5,  10% glycerol.  Store at 4 oC .

'''SP Elution Buffer''': 50 mM Phosphate Buffer, pH 7.5 , '''1.5 M''' NaCl,  100 mM arginine, 10% glycerol.  check pH to see if the buffer pH is ~8.0. Store at 4 oC .

'''Dialysis Buffer''': '''50% glycerol,''' 500 mM NaCl, 20 mM '''HEPES''',  pH 7.4, '''100 mM Arginine''', '''0.1 mM EDTA'''.   '''1 mM'''  DTT, Store at 4 oC or in cold room.

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

1. Transform  BL21 star (DE3) with pET28b-Cre (Clone # 374), plate on LB/Kan, grow on 37 oC o/n.

2, Pick a few well isolated colonies, grow in 200 ml LB/Kan with shaking overnight at 37 oC.  Make Glycerol stock. 

3. Make 20 L  LB-G medium

310 g 2xYT  powder

150g NaCl

40 g Peptone or tryptone

200 ml glycerol.

autoclave, use 8 L  for each purification.

4. Aliquote  the overnight culture in a ratio of 1:50 to 1 L  LB-G medium and incubate with vigorous shaking at 37 oC until an OD600 > 1, usually 2~4 hours

5. Induce Cre expression by adding 1 M IPTG solution to a final concentration of   1  mM (add  1 ml of 1 M IPTG to 1 L) and induce   2~4 hours at 37 oC, or 0.2 mM IPTG o/n at 25~ 30 oC.

 6. Spin down cells,  6 min at 6000 rpm, RT., transfer pellet to one centrifuge tube. Weigh wet pellet, record weigh onto protein database. Add 100 ml Lysis Buffer to pellet and store at -80 oC o/n.

7. Thaw cell pellet, add ~400 ml '''cold Lysis Buffer.'''

'''If cells are not lysed, do step 8 and 9.'''

8. add  freshly prepared lysozyme to final conc 1 mg/mL,  and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml).

9.  Incubate for  30 ~90 min with stiring at room temperature, or cold room (4 oC) overnight, until the cells are lysed, and DNA is digested

10  Spin again at 25,000 rpm for 35 min at 4 oC on ultracentrifuge.

If vaccum can not go down, or speed can not go up to > 3000 rpm

*stop centrifuge.
*Check if centrifuge tube is broken or missing a seal ring.
*Check if centrifuge caps are tighten

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

Rotor should be  cooled down using dry ice. Remove residual water with clean paper or cloth .

11. Resuspend pellet with 200 ml of Lysis Buffer. homogenize for 10 sec and spin at 30000 rpm again.

12. Combine supernatant and  load to a 50 -100 ml Ni  column.

13. Wash  column with 200~ 1000 ml '''Lysis Buffer '''until OD280 drops to baseline.

Wash column with 2 bed vol of 1.5 M NaCl, 50 mM K-PO4, pH 7.5 

14. Wash column with 1 CV of  '''Buffer A'''.

15. Elute protein with  200 ml or more of  '''Ni Elution Buffer. (350 ml)'''

'''16. Dilute eluted protein solution with 3 volume of ''' '''Cre Dilution Buffer (1000 ml). Watch solution carefully, if protein is aggregating (protein solution turns to turbid), add solid arginine and stire immediately until the solution turns to clear! '''

'''17. Load to a '''10 ml '''Q FF '''column (Pharmacia) connected to a column packed with 30~50 ml '''Capto  SP ImpRes''' or  '''SP FF or SP HP or 20 ml ''', preequilibrated with''' Cre Dilution Buffer. Q FF is required for removing endotoxin.'''

Dilute eluant with > 2 volume of SP Dilution Buffer, load to Q FF + SP FF columns using P1 pump + air bubble detector.

18. Wash '''Q FF + SP FF''' columns with '''Cre Dilution Buffer''' or '''SP Washing Buffer '''until OD280 reaches baseline, optionally , use FPLC to monitor washing.

'''optional: '''Disconnect Q FF.

19. Elute with 5~10 CV of  '''SP Elution Buffer  '''''by reverse flow (invert column)''''', '''collect 5 ml fractions, 

optionally, elute using linear gradient from  '''Cre Dilution Buffer to SP Elution Buffer, 10 CV'''

Cre will be eluted at ~ 0.8 M to 1.2 M NaCl at pH 7.5 ~8.0.

20.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml). 

Combine fractions containing high protein concentration

21. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  Check if the dialysis tube is leaking.

22. Transfer Cre protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT,  -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with  45~50 ml of '''Dialysis Buffer  '''(in 100 ml beaker) in fridge, '''mix occasionally for several times,''' then dialyse overnight to concentrate protein, Measure OD280. 

For freeze drying: use Dialysis Buffer A, otherwise use Dialysis B

23. Clean Q FF and SP FF columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water (for longterm storage, wash with 20% ethanol).

24. Next morning, change '''Dialysis Buffer, '''dialyse for additional 1~6 hours, mix occasionally 

25. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3 X 1 ml of '''1 M''' arginine, combine proteins.

26. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at 1~1.2  mg/vial.'''   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer''' as control to test buffer toxicity.

27. Put vials immediately on ice. Create a new lot number in the protein and product databases, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

38. Place the freezer box in a freezer box,    store the freezer box in -80 oC freezer.  

39. Wash Dialysis tube with water, store in  20% ethanol in a bottle container

'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''

== ==

== Old Protocols: ==

== Current Working Protocol for  Product in Liquid Form ==

'''Materials''' 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen). ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC. Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use. Bradford reagent (Sigma) with bovine serum albumin (BSA) standard. Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

'''Note:'''

'''Cre''': pI/Mw: 10.30 / 42764.07

*Add 0.05 mM EDTA and 1 mM beta-ME to all buffers
*'''Step 11 to 19 should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 25 and the yield will be very low if glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.
*Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

=== '''Buffers''' ===

'''Buffer Q''': 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 50 mM NaCl, 1 mM EDTA.

'''Buffer Q2''': 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 50 mM NaCl, 1 mM EDTA.

'''Suspension Buffer''': 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

'''Lysis Buffer''': 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol,  1 mM beta-ME.

'''Buffer A''', 25 mM Tris-HCl, pH 8.0,  500 mM NaCl, '''10 mM '''imidazole, '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' check pH ~8. Store at 4 oC or in cold room.

'''Buffer A2''', 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol. 1 mM beta-ME. Store at 4 oC or in cold room.

'''TT buffer''': 

tartaric salt buffer: 1.5 M L-Tartaric acid (Disodium salt) 50 mM phosphate buffer, pH 7.4 20 mM imidazole

Store at 25 oC

 '''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC or in cold room. '''Ni Elution buffer''':  50 mM Phosphate Buffer, '''pH 7.0''', '''250 mM NaCl''', '''100 mM arginine,'''   '''300 mM imidazole''',  10% glycerol. Store at 4 oC or in cold room.

'''Cre Dilution Buffer:''' 50 mM Phosphate Buffer,  pH 7.0,  1 mM beta-ME, '''0.1 mM EDTA'''. Store at 4 oC or in cold room.

 '''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.1 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Dilution Buffer'''''': '''50 mM Phosphate Buffer, pH 7.0,  0.1 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

'''SP Washing Buffer''': 50 mM Phosphate Buffer, pH '''7.0''', '''500 mM NaCl''',  '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Elution Buffer''': 50 mM Tris-HCl, pH '''8.0''', '''1.5 M''' NaCl,  5% glycerol,   '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use, check pH to see if the buffer pH is ~8.0. Store at 4 oC or in cold room.

'''Dialysis Buffer''': 50% glycerol, 500 mM NaCl, 20 mM '''HEPES''' or 50 mM '''Na-Phophate '''Buffer,  pH 7.4, 100 mM Arginine, '''0.1 mM EDTA'''. Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use Store at 4 oC or in cold room.

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

'''Expression and Purification'''

 1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid, for example, 1708 pT7A3-HTNCre

2. Make 8 L '''LB'''

3. Inoculate 200 ml LB medium supplemented with 0.5% (w/v) glucose, 50 ug/ml '''Kan''', and '''34 ug/ml chloramphenicol''' with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate  LB medium supplemented with 0.5% (w/v) '''glucose''',  50 ug/ml '''Kan''', and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

5. Induce expression by adding 1 M IPTG solution to a final concentration of  0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.

6. Spin down cells,  5 min at 6000 rpm, RT.  Add 100 ml Lysis Buffer to pellet and store at -80 oC. 7. Thaw cell pellet, add ~400 ml '''cold Lysis Buffer.'''

'''If cells are not lysed, do step 8 and 9.'''

8. add  freshly prepared lysozyme to final conc 1 mg/mL,  and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml). 9.  Incubate at room temperature for a few hours, or cold room overnight, until the cells are lysed.

10.  Add equal volume of '''TT buffer''.'''''

'''''The folling steps (11 to 19) should be completed in 1 working day.'''''

 '''''11. Homogenize or sonicate on ice, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more buffer'''''(equal vol mix of Buffer A and TT buffer)'''', '''then homogenize on ice again.

If it is still too viscous (sticky)

Add more  benzonase or DNase I  to the lysate and incubate for 30 min at room temperature. Sonicate the suspension on ice for 1 min (power 50%, cycles 5)

'''Note:'''

It was reported that sonication for 1 min with probe will cause Cre to form soluble aggregate. It is better to avoid homegenization and sonication. Use water bath-type of  mild sonicator is recommended after lysozyme and DNase 1 digestion:

30 s on/30 s off pulses, at low intensity for 15 minutes

Ref: [http://www.biomedcentral.com/1472-6750/9/80 http://www.biomedcentral.com/1472-6750/9/80] 12.  Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down:

*stop centrifuge.
*Check if centrifuge tube is broken or missing a seal ring.
*Check if centrifuge caps are tighten

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

Rotor should be stored in cold room or cool down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.

13. Resuspend pellet with 200 ml of Lysis Buffer + TT Buffer (100 ml '''Lysis Buffer''' + 100 ml '''TT buffer)'''. Homogenize and spin at 25000 rpm again.

14. Combine supernatant and  load to a 10 ~15 ml Ni  column.

15. Wash  column with 200~ 500 ml '''Buffer A '''until OD280 drops to baseline.

16. Wash column with 1 CV of  '''Buffer A2'''.

17. Elute protein with  150 ml or more of  '''Ni Elution Buffer (not Buffer B) directly to'''  10 ml '''Q FF '''column (Pharmacia) connected to a column packed with 30 ml '''SP FF''', preequilibrated with''' SP Dilution Buffer. Q FF is required for removing endotoxin.'''

18. Wash Q FF + SP FF columns with '''SP Dilution Buffer''' until OD280 reaches baseline, optionally , use FPLC to monitor washing.

19. '''Disconnect Q FF,''' Wash SP FF with 2~10 CV of '''SP Washing Buffer''' until OD280 reaches baseline, optionally , use FPLC to monitor washing.

20. Elute with 5~10 CV of  '''SP Elution Buffer, '''collect 10 ml fractions

Cre will be eluted at ~ 0.8 M to 1.2 M NaCl at pH 7.5 ~8.0.

21.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).  Combine  fractions containing low protein concentration and concentrate proteins to ~2  to  5  mg using Pierce 9k spin columns. Combine fractions containing high protein concentration and spin-column concentrated protein solution.

22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  Check if the dialysis tube is leaking.

23. Transfer Cre protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT,  -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with  45~50 ml of '''Dialysis Buffer  '''(in 100 ml beaker) overnight to concentrate protein, Measure OD280.

24. Clean Q FF and SP FF columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

25. Change dialysis buffer and dialyse for another 8 hours or o/n

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 2 X 0.5 ml of '''1 M''' arginine, combine proteins.

27. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2  mg/vial.'''   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.

28. Put vials immediately on ice. Create a new lot number in the protein and product databases, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

29. Store the HTNCre vials in a freezer box at −20 oC o/n.

30. Place the freezer box in a foam box, put the foam box in −80 oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''

'''Materials''' 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen) without His tag or DNAse I. ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC. <s>Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use.</s> <s>Bradford reagent (Sigma) with bovine serum albumin (BSA) standard.</s> Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

'''Note:'''

'''Cre''': pI/Mw: 10.30 / 42764.07

*Add 0.001 mM  (1 uM) EDTA and 1 mM beta-ME to all buffers
*'''Step 11 to 19 should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 25 and the yield will be very low if glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.
*<s>Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.</s>

=== '''Buffers''' ===

'''Buffer Q''': 25 mM Tris-HCl, pH 8.0, 1 mM beta-ME, 10% glycerol, 50 mM NaCl, 0.001 mM EDTA.

'''Buffer Q2''': 25 mM Tris-HCl, pH 8.0, 1 mM beta-ME, 50 mM NaCl, 0.001 mM EDTA.

'''Suspension Buffer''': 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 10% glycerol, 0.001 mM EDTA, 1 mM beta-ME.

'''Lysis Buffer''': 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, 5% glycerol,  1 mM beta-ME.

'''Buffer A''', 25 mM Tris-HCl, pH 8.0,  500 mM NaCl, '''10 mM '''imidazole, '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' check pH ~8. Store at 4 oC or in cold room.

'''Buffer A2''', 25 mM Tris-HCl, pH 8.0, 10 mM imidazole, 5% glycerol. 1 mM beta-ME. Store at 4 oC or in cold room.

'''TT buffer''': 

tartaric salt buffer: 1.5 M L-Tartaric acid (Disodium salt) 50 mM phosphate buffer, pH 7.4 20 mM imidazole

Store at 25 oC

 '''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC or in cold room. '''Ni Elution buffer''':  50 mM Phosphate Buffer, '''pH 7.0''', '''100 mM NaCl''', '''100 mM arginine,'''   '''150 mM imidazole''',  '''10% glycerol'''. Store at 4 oC or in cold room.

'''Cre Dilution Buffer:''' 50 mM Phosphate Buffer,  pH 7.0,  1 mM beta-ME, '''0.001 mM EDTA'''. Store at 4 oC or in cold room.

 '''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.001 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Dilution Buffer'''''': '''50 mM Phosphate Buffer, pH 7.0,  0.001 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

'''SP Washing Buffer '''(No glycerol): 50 mM Phosphate Buffer, pH '''7.0''', '''200 mM NaCl''',  '''0.001 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Elution Buffer''' (No glycerol): 50 mM Tris-HCl, pH '''8.'''0, '''1.5 M NaCl,  100 mM arginine, 15% Trehalose, ''' '''0.001 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use, check pH to see if the buffer pH is ~8.0. Store at 4 oC or in cold room.

'''Dialysis Buffer A '''(No glycerol): 500 mM NaCl, 10 mM '''HEPES''',  pH 7.4, 100 mM Arginine,  15% Trehalose, 0.001 mM EDTA, add 1 mM DTT''' just before use. Store at 4 oC or in cold room.'''

'''Dialysis Buffer B '''(with glycerol): 500 mM NaCl, 10 mM '''HEPES''',  pH 7.4, 100 mM Arginine,  50% glycerol, 0.001 mM EDTA, add 1 mM DTT''' just before use. Store at 4 oC or in cold room.'''

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

'''Expression and Purification'''

 1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid, for example, 1708 pT7A3-HTNCre or 1010 pBad-Cre

2. Make 20 L  LB Plus medium

500 g LB powder

200 g yeast extract

80 g Na2HPO4.7H2O

10 g KH2PO4

40 g glucose

autoclave, use 8 L  for each purification.

3. Inoculate 200 ml   LB Plus  medium supplemented with   50 ug/ml '''Kan (for 1708) or 100 ug/ml Amp (for 1010) '''and '''34 ug/ml chloramphenicol''' with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate  LB Plus medium supplemented with    50 ug/ml '''Kan  (for 1708) or 100 ug/ml Amp (for 1010) ''' and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600 > 0.4

5. Induce Cre expression by adding 1 M IPTG solution to a final concentration of  0.2 mM (add 0.2  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.---> for clone 1708

or  Induce expression by adding 1~2 g arabinose to 1 L   and induce  o/n at 30 oC,  or 6~8 hours at 37 oC---> for clone 1010

6. Spin down cells,  5 min at 6000 rpm, RT., transfer pellet to one centrifuge tube. Weigh wet pellet, record weigh onto protein database. Add 100 ml Lysis Buffer to pellet and store at -80 oC o/n.

7. Thaw cell pellet, add ~400 ml '''cold Lysis Buffer.'''

'''If cells are not lysed, do step 8 and 9.'''

8. add  freshly prepared lysozyme to final conc 1 mg/mL,  and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml).

9.  Incubate at room temperature for a few hours, or cold room overnight, until the cells are lysed, and DNA is digested

10.  Add equal volume of '''TT buffer'' (optional)'''''

'''''The folling steps (11 to 19) should be completed in 1 working day.'''''

 '''''11. Homogenize <s>or sonicate </s>on ice, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more buffer'''''(equal vol mix of Buffer A and TT buffer)''', '''then homogenize on ice again.

If it is still too viscous (sticky)

Add more  benzonase (no his tag) or DNase I  to the lysate and incubate for 30 ~90 min at room temperature. Sonicate the suspension on ice for 1 min (power 50%, cycles 5)

'''Note:'''

It was reported that '''sonication for 1 min with probe will cause Cre to form soluble aggregate.''' It is better to avoid homegenization and sonication. Use water bath-type of  mild sonicator is recommended after '''lysozyme and DNase 1''' digestion:

30 s on/30 s off pulses, at low intensity for 15 minutes

Ref: [http://www.biomedcentral.com/1472-6750/9/80 http://www.biomedcentral.com/1472-6750/9/80] 12.  Spin again at 30,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down, or speed can not go up to > 3000 rpm

*stop centrifuge.
*Check if centrifuge tube is broken or missing a seal ring.
*Check if centrifuge caps are tighten

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

Rotor should be stored in cold room or cool down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.

<s>13. Resuspend pellet with 200 ml of Lysis Buffer + TT Buffer (100 ml '''Lysis Buffer''' + 100 ml '''TT buffer)'''. Homogenize and spin at 25000 rpm again.</s>

13. Resuspend pellet with 200 ml of Lysis Buffer . Homogenize for 10 sec and spin at 30000 rpm again.

14. Combine supernatant and  load to a 50 ml Ni  column.

15. Wash  column with 200~ 1000 ml '''Buffer A '''until OD280 drops to baseline.

16. Wash column with 1 CV of  '''Buffer A2'''.

17. Elute protein with  150 ml or more of  '''Ni Elution Buffer (not Buffer B) directly to'''  10 ml '''Q FF '''column (Pharmacia) connected to a column packed with 30~50 ml '''SP FF''', preequilibrated with''' SP Dilution Buffer. Q FF is required for removing endotoxin.'''

'''Optionally, '''elute Cre into 10 ml fractions with '''Ni Elution Buffer''', wach the color of eluant while the protein is eluting out from column,  if you see protein is aggregating (solution is becoming turbid), add  a few ml of 1 M arginine and shake gently until solution is clear.  Dilute eluant with 2 volume of SP Dilution Buffer, load to Q FF + SP FF columns using P1 pump + air bubble detector.

18. Wash '''Q FF + SP FF''' columns with '''SP Dilution Buffer''' until OD280 reaches baseline, optionally , use FPLC to monitor washing.

19. Wash Q FF+ SP FF with 2 CV of '''SP Washing Buffer''' until OD280 reaches baseline,   use FPLC to monitor washing. 

'''optional: '''Disconnect Q FF.

20. Elute with 5~10 CV of  '''SP Elution Buffer ''''''by reverse flow'''''', '''collect 10 ml fractions, 

optionally, elute using linear gradient from  '''SP Washing Buffer to SP Elution Buffer, 10 CV'''

Cre will be eluted at ~ 0.8 M to 1.2 M NaCl at pH 7.5 ~8.0.

21.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml). 

optionally, combine  fractions containing low protein concentration and concentrate proteins to ~2  to  5  mg using Pierce 9k spin columns.

Combine fractions containing high protein concentration and spin-column concentrated protein solution.

'''Optional''': Dilute combined fractions with 10 volumes of SP Dilution Buffer. Load to 10~20 ml heparin sepharose column. Wash extensively with SP Dilution Buffer. Then elute Cre using linear gradient from SP Dilution Buffer to SP Elution Buffe'''r'''<u

22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  Check if the dialysis tube is leaking.

23. Transfer Cre protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT,  -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with  45~50 ml of '''Dialysis Buffer  '''(in 100 ml beaker) in fridge, '''mix occasionally for several times,''' then dialyse overnight to concentrate protein, Measure OD280. 

For freeze drying: use Dialysis Buffer A, otherwise use Dialysis B

24. Clean Q FF and SP FF columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water (for longterm storage, wash with 20% ethanol).

25. Next morning, change '''Dialysis Buffer, '''dialyse for additional 3~6 hours, mix occasionally 

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3 X 1 ml of '''1 M''' arginine, combine proteins.

27. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2  mg/vial.'''   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer''' as control to test buffer toxicity.

28. Put vials immediately on ice. Create a new lot number in the protein and product databases, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

29. Store the HTNCre vials in a freezer box at −20 oC o/n.

30. Place the freezer box in a foam box, put the foam box in −80 oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''

== ==

== Old Protocols: ==

== Current Working Protocol for  Product in Liquid Form ==

'''Materials''' 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen). ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC. Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use. Bradford reagent (Sigma) with bovine serum albumin (BSA) standard. Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

'''Note:'''

'''Cre''': pI/Mw: 10.30 / 42764.07

*Add 0.05 mM EDTA and 1 mM beta-ME to all buffers
*'''Step 11 to 19 should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 25 and the yield will be very low if glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.
*Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

=== '''Buffers''' ===

'''Buffer Q''': 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 50 mM NaCl, 1 mM EDTA.

'''Buffer Q2''': 25 mM Tris-HCl, pH 8.0, 1 mM b-ME, 50 mM NaCl, 1 mM EDTA.

'''Suspension Buffer''': 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

'''Lysis Buffer''': 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol,  1 mM beta-ME.

'''Buffer A''', 25 mM Tris-HCl, pH 8.0,  500 mM NaCl, '''10 mM '''imidazole, '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' check pH ~8. Store at 4 oC or in cold room.

'''Buffer A2''', 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol. 1 mM beta-ME. Store at 4 oC or in cold room.

'''TT buffer''': 

tartaric salt buffer: 1.5 M L-Tartaric acid (Disodium salt) 50 mM phosphate buffer, pH 7.4 20 mM imidazole

Store at 25 oC

 '''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC or in cold room. '''Ni Elution buffer''':  50 mM Phosphate Buffer, '''pH 7.0''', '''250 mM NaCl''', '''100 mM arginine,'''   '''300 mM imidazole''',  10% glycerol. Store at 4 oC or in cold room.

'''Cre Dilution Buffer:''' 50 mM Phosphate Buffer,  pH 7.0,  1 mM beta-ME, '''0.1 mM EDTA'''. Store at 4 oC or in cold room.

 '''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.1 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Dilution Buffer'''''': '''50 mM Phosphate Buffer, pH 7.0,  0.1 mM EDTA, 100 mM arginine, 10% glycerol, 1 mM beta-ME.

'''SP Washing Buffer''': 50 mM Phosphate Buffer, pH '''7.0''', '''500 mM NaCl''',  '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Elution Buffer''': 50 mM Tris-HCl, pH '''8.0''', '''1.5 M''' NaCl,  5% glycerol,   '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use, check pH to see if the buffer pH is ~8.0. Store at 4 oC or in cold room.

'''Dialysis Buffer''': 50% glycerol, 500 mM NaCl, 20 mM '''HEPES''' or 50 mM '''Na-Phophate '''Buffer,  pH 7.4, 100 mM Arginine, '''0.1 mM EDTA'''. Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use Store at 4 oC or in cold room.

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

'''Expression and Purification'''

 1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid, for example, 1708 pT7A3-HTNCre

2. Make 8 L '''LB'''

3. Inoculate 200 ml LB medium supplemented with 0.5% (w/v) glucose, 50 ug/ml '''Kan''', and '''34 ug/ml chloramphenicol''' with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate  LB medium supplemented with 0.5% (w/v) '''glucose''',  50 ug/ml '''Kan''', and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

5. Induce expression by adding 1 M IPTG solution to a final concentration of  0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.

6. Spin down cells,  5 min at 6000 rpm, RT.  Add 100 ml Lysis Buffer to pellet and store at -80 oC. 7. Thaw cell pellet, add ~400 ml '''cold Lysis Buffer.'''

'''If cells are not lysed, do step 8 and 9.'''

8. add  freshly prepared lysozyme to final conc 1 mg/mL,  and DNAse I (100 μg/mL) or benzonase (final conc 20 U/ml). 9.  Incubate at room temperature for a few hours, or cold room overnight, until the cells are lysed.

10.  Add equal volume of '''TT buffer''.'''''

'''''The folling steps (11 to 19) should be completed in 1 working day.'''''

 '''''11. Homogenize or sonicate on ice, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more buffer'''''(equal vol mix of Buffer A and TT buffer)'''', '''then homogenize on ice again.

If it is still too viscous (sticky)

Add more  benzonase or DNase I  to the lysate and incubate for 30 min at room temperature. Sonicate the suspension on ice for 1 min (power 50%, cycles 5)

'''Note:'''

It was reported that sonication for 1 min with probe will cause Cre to form soluble aggregate. It is better to avoid homegenization and sonication. Use water bath-type of  mild sonicator is recommended after lysozyme and DNase 1 digestion:

30 s on/30 s off pulses, at low intensity for 15 minutes

Ref: [http://www.biomedcentral.com/1472-6750/9/80 http://www.biomedcentral.com/1472-6750/9/80] 12.  Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down:

*stop centrifuge.
*Check if centrifuge tube is broken or missing a seal ring.
*Check if centrifuge caps are tighten

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

Rotor should be stored in cold room or cool down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.

13. Resuspend pellet with 200 ml of Lysis Buffer + TT Buffer (100 ml '''Lysis Buffer''' + 100 ml '''TT buffer)'''. Homogenize and spin at 25000 rpm again.

14. Combine supernatant and  load to a 10 ~15 ml Ni  column.

15. Wash  column with 200~ 500 ml '''Buffer A '''until OD280 drops to baseline.

16. Wash column with 1 CV of  '''Buffer A2'''.

17. Elute protein with  150 ml or more of  '''Ni Elution Buffer (not Buffer B) directly to'''  10 ml '''Q FF '''column (Pharmacia) connected to a column packed with 30 ml '''SP FF''', preequilibrated with''' SP Dilution Buffer. Q FF is required for removing endotoxin.'''

18. Wash Q FF + SP FF columns with '''SP Dilution Buffer''' until OD280 reaches baseline, optionally , use FPLC to monitor washing.

19. '''Disconnect Q FF,''' Wash SP FF with 2~10 CV of '''SP Washing Buffer''' until OD280 reaches baseline, optionally , use FPLC to monitor washing.

20. Elute with 5~10 CV of  '''SP Elution Buffer, '''collect 10 ml fractions

Cre will be eluted at ~ 0.8 M to 1.2 M NaCl at pH 7.5 ~8.0.

21.   Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).  Combine  fractions containing low protein concentration and concentrate proteins to ~2  to  5  mg using Pierce 9k spin columns. Combine fractions containing high protein concentration and spin-column concentrated protein solution.

22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  Check if the dialysis tube is leaking.

23. Transfer Cre protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT,  -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with  45~50 ml of '''Dialysis Buffer  '''(in 100 ml beaker) overnight to concentrate protein, Measure OD280.

24. Clean Q FF and SP FF columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

25. Change dialysis buffer and dialyse for another 8 hours or o/n

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 2 X 0.5 ml of '''1 M''' arginine, combine proteins.

27. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2  mg/vial.'''   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.

28. Put vials immediately on ice. Create a new lot number in the protein and product databases, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

29. Store the HTNCre vials in a freezer box at −20 oC o/n.

30. Place the freezer box in a foam box, put the foam box in −80 oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''

== Purification of Cre using HiTrap Heparin  ==

To prepare 1 M K-Phosphate: 300 ml of 1 M KH2PO4, 700 ml 1M K2HPO4, the pH is 7.0 measured using a pH meter.

'''Buffer A''': To make 20 mM K-Phophate, 5% glycerol: 40 ml 1 M K-Phosphate Buffer, pH 7.0, 100 ml glycerol, add ddH2O to 2 L.

'''Buffer B''': To make  20 mM K-Phophate, 1.5 M NaCl, 5% glycerol: dissolve 87.66 g NaCl in H2O,  40 ml 1 M K-Phosphate Buffer, pH 7.0, 100 ml glycerol, add ddH2O to 2 L

'''Column''': 1 ml column, equilibrate with 20 mM Potassium Phosphate Buffer, pH 7.0.

Load 150 ml sample containing ~100 mM KCl (1:10 dilution from  sample eluted from SP FF using 1 M KCl, pH 7.0).

Linear gradient 15 ml from Buffer A to B, 1.5 ml/min, 3 ml/fraction. OD260 nanodrop: 0.8 mg/ml, total 2.4 mg in fraction 6.

Look like Cre is eluted at 0.7 M NaCl, when pH is 7.0

Date:5/19/2019, by Paul Xu

[[File:1ml heparin Cre.JPG|RTENOTITLE]]

== ==

== Purification of Cre using SOURCE 30S ==

'''30S-D'''

2.3 g dibasic NaPO4, 2.6 g monobasic KPO4 or 40 mM Sodium Phosphate (mix 102 ml of 0.2 M NaH2PO4 and 98 ml of 0.2 M Na2HPO4) 15 g NaCl, 0.25 M 50g glycerol (check pH to see if it is ~ 6.8) Fill to 1 L with diH2O

'''30S-W'''

2.3 g dibasic NaPO4, 2.6 g monobasic KPO4 or 40 mM Sodium Phosphate (mix 102 ml of 0.2 M NaH2PO4 and 98 ml of 0.2 M Na2HPO4) 30 g NaCl, 0.5 M 50g glycerol (check pH to see if it is ~ 6.8) Fill to 1 L with diH2O

 '''30S-E''' 1.2 g dibasic NaPO + 1.3 g monobasic KPO4 or 40 mM Sodium Phosphate (mix 102 ml of 0.2 M NaH2PO4 and 98 ml of 0.2 M Na2HPO4) 88 g NaCl, 1.5 M 25 g glycerol (check pH to see if it is ~ 6.8) Fill with diH2O to 1 L

1. Mix protein solution eluted from Ni column with equal volume of Buffer 30S-D.

2. Load protein to 15 ml SOURCE 30S column, pre-equilibrated with Buffer 30S-W.

3. Wash column with 30S-W until no protein is detectable (OD280 <0.05).

4. Elute Tat-Cre with Buffer '''30S-E'''

== '''Previous Working Protocol''' ==

'''Materials''' 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen). ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC. Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use. Bradford reagent (Sigma) with bovine serum albumin (BSA) standard. Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

'''Note:'''

'''Cre''': pI/Mw: 10.30 / 42764.07

*Add 0.05 mM EDTA and 1 mM beta-ME to all buffers
*'''Step 7 to 24 should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 11 and the yield will be very low if TT buffer/glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.
*Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

 '''Buffers'''

'''Buffer Q''': 50 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 1 mM EDTA.

'''Buffer Q2''': 50 mM Tris-HCl, pH 8.0, 1 mM b-ME, 1 mM EDTA.

'''Suspension Buffer''': 50 mM Phosphate Buffer, pH 7.4,   500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

'''Buffer A''', 50 mM Phosphate Buffer, pH 7.4, 600 mM NaCl, '''20 mM '''imidazole, '''0.05 mM''' EDTA,   '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' Store at 4 oC or in cold room.

'''4 L:'''lute Cre pro

190 ml of 0.2 M NaH2PO4 (Dilute from 2 M NaH2PO4)

810 ml of 0.2 M Na2HPO4  (dilute from 0.5 M Na2HPO4)

140 g NaCl

2.7 g Imidazole

2 ml 0.1 M EDTA

dd water to 4 L

Before use: add 70 ul beta-ME to 1 L

 '''TT buffer''': 2M  disodium tartrate. Store at 25 oC '''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC or in cold room. '''Ni Elution buffer''':  50 mM Phosphate Buffer, pH 7.4, 150 mM NaCl, 20 mM imidazole, 0.05 mM EDTA,  250 mM imidazole, 250 mM Arginine, 10% glycerol. Store at 4 oC or in cold room.

'''Cre Dilution Buffer:''' 50 mM Phosphate Buffer,  pH 7.4,  1 mM beta-ME, '''0.1 mM EDTA'''. Store at 4 oC or in cold room.

'''SP Washing Buffer''': 50 mM Phosphate Buffer, pH 7.4, 500 mM NaCl,  '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.1 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Elution Buffer''': 50 mM Phosphate Buffer, pH 7.4, 1 M NaCl,  250 mM Arginine,  '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''MEP Equilibration Buffer''': 20 mM Phosphate Buffer, pH 7.4, 1M NaCl, 50 mM Arginine. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

'''MEP Washing Buffer''': 20 mM Phosphate Buffer, pH 6.0,  Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

'''MEP Elution Buffer''': 50 mM NaOAc, pH 5.0, '''300 mM''' Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

 '''Dialysis Buffer''': 50% glycerol, 500 mM NaCl, 20 mM '''HEPES''' or 50 mM '''Na-Phophate '''Buffer,  pH 7.4, 100 mM Arginine, '''0.1 mM EDTA'''. Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use Store at 4 oC or in cold room.

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

'''Expression and Purification'''

 1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid 1708 pT7A3-HTNCre

2. Make 7 L '''LB'''

3. Inoculate 200 ml LB medium supplemented with 1% (w/v) glucose, 50 ug/ml '''Kan''', and '''34 ug/ml chloramphenicol''' with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate <strike>TB or</strike> LB medium supplemented with 1% (w/v) '''glucose''',  50 ug/ml '''Kan''', and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

5. Induce expression by adding 1 M IPTG solution to a final concentration of <strike>0.3 to 0.5</strike> 0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.

6. Pellet cells by centrifugation (6000 rpm, 4 min) and freeze the bacterial pellets at −20 oC, or -80 oC, or dry ice. The cell pellets can be stored at −20 oC for several days, but it is advisable to use them as soon as possible.

7. Thaw pellets in warm water and resuspend in  '''15 ml Buffer Q per liter culture '''using a magnetic plastic stick. Repeat freeze-thawing if cells are not lysed after one freezing-thawing.

8. Homogenize for 1  min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more equal vol mix of '''Buffer Q'''  if it is still sticky, optioally add 25 U/ml benzonase or DNase I to the suspension and incubate for 30 min at room temperature. Sonicate the suspension on ice for 2 min (power 50%, cycles 5) if it is still sticky.

 9. Centrifuge the lysate for 20 min at 4 oC and  12000 rpm (do not run > 12000 rpm).

Spin again at 25,000 rpm for 25 min at 4 oC on ultracentrifuge. Check if any one of the centrifuge tubes is broken if vaccum can not go down. If one of the tubes is broken, or seal is missing or cap is not tighten, vaccum will not go down and the centrifuge vaccum pump will be damaged by evaporated salt and water.

Make sure ultracentrifuge tubes are not broken, tighten tubes before placing them into rotor. Rotor should be stored in cold room or cool down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.

10. Decant the supernatant, load to a QFF (2.5×40 cm XK25) column couple to a SP FF (2.5×20 cm,  XK25, 30~50 ml)  column.

11. Wash QFF/SP FF columns with 500 to 1000 ml '''Buffer Q2 '''until OD280 drop to baseline.

12. Disconnect QFF. Wash SP FF column with '''SP Washing Buffer''' until OD280 drop to baseline.

13. Elute protein '''directly''' to 15~ 20 ml Ni column (EDTA compatible resin from Roche) using 150~ 200 ml of '''SP Elution Buffer '''at flow rate 1~3 ml/min.

Cre will be eluted at ~ 0.8 M NaCl at pH 7.5.

14. Wash column with 10 CV of Buffer A.

'''Optional: '''remove Endotoxin before elution:

Wash column with 100 ml of ETR Buffer to remove endotoxin

or

Wash the Ni beads 3 times with 2 CV of 0.1 M Arginine, or 2 times with 2 CV of 0.5 M Arginine.

After step 14,  clean Q FF and SP FF columns with 2 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

'''Optional steps 15 to 17'''

15. Elute Cre protein directly into a 15 ml MEP column by reverse-flow using '''Ni Elution buffer.'''

Ni column can be regenerated using EDTA, Urea, and then NiCl2.

Cre will bind to MEP at 1 M NaCl.

16. Wash MEP column with '''MEP Washing Buffer'''

17. Elute Cre protein by reverse-flow using '''MEP Elution Buffer'''. Collect 5 ml fraction. Combine fractions containing Cre protein.  Measure protein using nanodrop.

Cre can be eluted from MEP column at pH 5.0, 300 mM Arginine.

MEP column can be regernated using 0.1 N Acetic acid, water, 0.1 N NaCl.

18. Elute protein using'''Ni Elution buffer.''' Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).  Concentrate protein to ~ 5 to 10 mg using Pierce 9k spin columns.

19. Wash a dialysis tube thoroughly with ddH2O,  Check if the dialysis tube is leaking.

20. Transfer Cre protein solution to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT, top cube of 4 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein in  70 ml of '''Dialysis Buffer  '''(in 100 ml beaker) overnight to concentrate protein, Measure OD280.

21. Wash dialysis tube with 2 X 0.5 ml of '''1 M''' arginine, combine proteins.

22. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2  mg/vial.'''   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.

23. Put vials immediately on ice.  Make sure the protein is '''at least 1 mg per vial''' (give 1.2 mg so customers will not complain). Create a new lot number in the product database, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

24. Store the HTNCre vials in a freezer box at −20 oC o/n.

25. Place the freezer box in a foam box, put the foam box in −80 oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''

== '''Protocol 1''' ==

'''Materials''' 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen). ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC. Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use. Bradford reagent (Sigma) with bovine serum albumin (BSA) standard. Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

'''Note:'''

*Use Phosphate Buffer for purification. Do not use buffer closes to PI (pH>8).
*Add 0.05 mM EDTA and 1 mM beta-ME to all buffers
*'''Step 7 to 24 should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 11 and the yield will be very low if TT buffer/glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.
*Do not freeze protein at -80 oC immediately after dialysis. Store at -20 oC overnight then store at -80 oC.

 '''Buffers'''

'''Suspension Buffer''': 50 mM Phosphate Buffer, pH 7.4,   500 mM NaCl, 10% glycerol, 0.05 mM EDTA, 1 mM beta-ME.

'''Buffer Q''': 50 mM Tris-HCl, pH 8.0, 1 mM b-ME, 10% glycerol, 1 mM EDTA.

 '''Buffer A''', 50 mM Phosphate Buffer, pH 7.4, 600 mM NaCl, '''20 mM '''imidazole, '''0.05 mM''' EDTA,   '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' Store at 4 oC or in cold room.

'''4 L:'''lute Cre pro

190 ml of 0.2 M NaH2PO4 (Dilute from 2 M NaH2PO4)

810 ml of 0.2 M Na2HPO4  (dilute from 0.5 M Na2HPO4)

140 g NaCl

2.7 g Imidazole

2 ml 0.1 M EDTA

dd water to 4 L

Before use: add 70 ul beta-ME to 1 L

 '''TT buffer''': 2M  disodium tartrate. Store at 25 oC '''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC or in cold room. '''Ni Elution buffer''':  50 mM Phosphate Buffer, pH 7.4, 150 mM NaCl, 20 mM imidazole, 0.05 mM EDTA,  250 mM imidazole, 250 mM Arginine, 10% glycerol. Store at 4 oC or in cold room.

'''Cre Dilution Buffer:''' 50 mM Phosphate Buffer,  pH 7.4,  1 mM beta-ME, '''0.1 mM EDTA'''. Store at 4 oC or in cold room.

'''SP Washing Buffer''': 50 mM Phosphate Buffer, pH 7.4, 500 mM NaCl,  '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.1 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Elution Buffer''': 50 mM Phosphate Buffer, pH 7.4, 1 M NaCl,  250 mM Arginine,  '''0.1 mM EDTA'''.  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''MEP Equilibration Buffer''': 20 mM Phosphate Buffer, pH 7.4, 1M NaCl, 50 mM Arginine. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

'''MEP Washing Buffer''': 20 mM Phosphate Buffer, pH 6.0,  Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

'''MEP Elution Buffer''': 50 mM NaOAc, pH 5.0, '''300 mM''' Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

 '''Dialysis Buffer''': 50% glycerol, 500 mM NaCl, 20 mM '''HEPES''' or 50 mM '''Na-Phophate '''Buffer,  pH 7.4, 100 mM Arginine, '''0.1 mM EDTA'''. Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use Store at 4 oC or in cold room.

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

'''Expression and Purification'''

 1. Transform  Nico21(DE3)/plysN with the Cre expression plasmid 1708 pT7A3-HTNCre

2. Make 7 L '''LB'''

3. Inoculate 200 ml LB medium supplemented with 1% (w/v) glucose, 50 ug/ml '''Kan''', and '''34 ug/ml chloramphenicol''' with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate <strike>TB or</strike> LB medium supplemented with 1% (w/v) '''glucose''',  50 ug/ml '''Kan''', and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

5. Induce expression by adding 1 M IPTG solution to a final concentration of <strike>0.3 to 0.5</strike> 0.1 mM (add 0.1  ml of 1 M IPTG to 1 L) and induce  o/n at 25~ 30 oC.

6. Pellet cells by centrifugation (6000 rpm, 4 min) and freeze the bacterial pellets at −20 oC, or -80 oC, or dry ice. The cell pellets can be stored at −20 oC for several days, but it is advisable to use them as soon as possible.

7. Thaw pellets in warm water and resuspend in  '''15 ml Suspension Buffer per liter culture '''using a magnetic plastic stick. Repeat freeze-thawing if cells are not lysed after one freezing-thawing. Add equal volume of  cold TT Buffer.

8. Homogenize for 1  min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more equal vol mix of Suspension Buffer and TT buffer if it is still sticky, optioally add 25 U/ml benzonase or DNase I to the suspension and incubate for 30 min at room temperature. Sonicate the suspension on ice for 2 min (power 50%, cycles 5) if it is still sticky.

 9. Centrifuge the lysate for 20 min at 4 oC and  12000 rpm (do not run > 12000 rpm).

Spin again at 25,000 rpm for 25 min at 4 oC on ultracentrifuge. Check if any one of the centrifuge tubes is broken if vaccum can not go down. If one of the tubes is broken, or seal is missing or cap is not tighten, vaccum will not go down and the centrifuge vaccum pump will be damaged by evaporated salt and water.

Make sure ultracentrifuge tubes are not broken, tighten tubes before placing them into rotor. Rotor should be stored in cold room or cool down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.

10. Decant the supernatant, load to  30~50 ml SP FF column.

11. Wash SP FF column with 200~300 ml '''SP Washing Buffer'''

12. Elute protein '''directly''' to 15~ 20 ml Ni column (EDTA compatible resin from Roche) using 150~ 200 ml of '''SP Elution Buffer '''at flow rate 1~3 ml/min.

Cre will be eluted at ~ 0.8 M NaCl at pH 7.5.

13. Wash column with 10 CV of Buffer A.

14. Wash column with 100 ml of ETR Buffer to remove endotoxin.

'''Optional: '''remove Endotoxin before elution:

Wash the Ni beads 3 times with 2 CV of 0.1 M Arginine, or 2 times with 2 CV of 0.5 M Arginine.

After step 14,  clean SP column with 2 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

'''Optional steps 15 to 17'''

15. Elute Cre protein directly into a 15 ml MEP column by reverse-flow using '''Ni Elution buffer.'''

Ni column can be regenerated using EDTA, Urea, and then NiCl2.

Cre will bind to MEP at 1 M NaCl.

16. Wash MEP column with '''MEP Washing Buffer'''

17. Elute Cre protein by reverse-flow using '''MEP Elution Buffer'''. Collect 5 ml fraction. Combine fractions containing Cre protein.  Measure protein using nanodrop.

Cre can be eluted from MEP column at pH 5.0, 300 mM Arginine.

MEP column can be regernated using 0.1 N Acetic acid, water, 0.1 N NaCl.

18. Elute protein using'''Ni Elution buffer.''' Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).  Concentrate protein to ~ 5 to 10 mg using Pierce 9k spin columns.

19. Wash a dialysis tube thoroughly with ddH2O  Check if the dialysis tube is leaking.

20. Transfer Cre protein solution to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT, top cube of 4 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein in  70 ml of '''Dialysis Buffer  '''(in 100 ml beaker) overnight to concentrate protein, Measure OD280.

21. Wash dialysis tube with 2 X 0.5 ml of '''1 M''' arginine, combine proteins.

22. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   '''Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2  mg/vial.'''   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.

23. Put vials immediately on ice.  Make sure the protein is '''at least 1 mg per vial''' (give 1.2 mg so customers will not complain). Create a new lot number in the product database, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

24. Store the HTNCre vials in a freezer box at −20 oC o/n.

25. Place the freezer box in a foam box, put the foam box in −80 oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

'''Note: Do not use trehalose as it will promote protein aggregation. Use 1 M Arginine.'''

== Activity Assay ==

1. Transfect 203T cells in 24 well plate with reporer plasmid (0.5~1 ug)

 1522 CMV-Brainbow-2.1-R Plasmid (from Green to Red)

 2181: pCMV-LoxP-kozak-TurboRFP-ODC-2Stops-LoxP-TurboGFP (from Red to Green)

2. 12 hours after transfection, add Cre recombinase (sterize).

3. Take pictures under fluroencese microscope for 3~5 days, take a note of cell viability.

DO NOT USE

1. Convert protein concentration in mg/ml to molar concentration

Use this link: [http://www.my-whiteboard.com/pub/html/mw_2_moles.html http://www.my-whiteboard.com/pub/html/mw_2_moles.html]

2. Add Cre to LoxP reporter cell line (either stable cell line or 293T or 293E cells transfected with LoxP-GFP-LoxP-RFP) grown in  24 well plate, concentration 1, 2.5, 5, 7.5 and 10 uM.

3. Observe recombination efficiency using flourescent microscope.

== '''Protocol 2''' ==

'''Materials''' 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen). ready-to-use dialysis tubing is prepared according to the manufacturer’s instructions and stored in 50% ethanol at 4 oC. Lysozyme solution: dissolve lysozyme (Sigma) in 1X buffer A to a concentration of 80 mg/ml. Always prepare freshly before use. Bradford reagent (Sigma) with bovine serum albumin (BSA) standard. Low protein binding filter (e.g., Millex GV, 0 22 m, Millipore).

'''Note:'''

*Use Phosphate Buffer for purification. Do not use extreme buffer (pH>8).
*Step 7 to 17 should be completed within reasonable short time (Must be completed within a working day, can not stop in the middle)
*Cre protein rapidly form aggreggate from 43 kDa to very large MW. Large aggregate will be precipitated in Step 11 and the yield will be very low if TT buffer/glycerol and arginine is not added
*Do not handle protein at room temperature. Cre will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength (<100 mM NaCl) for a long time. Add arginine up to 0.5 M if low salt buffer is needed for purification.

 '''Buffers''' Buffer A, 50 mM Phosphate Buffer, pH 7.4, 500 mM NaCl, 10 mM imidazole,10% glycerol (adjust to 10% glycerol using used Dialysis Buffer II), 50 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. TT buffer: 500 mM disodium tartrate. Ni Washing buffer: the same as Buffer A. Ni Elution buffer:  Buffer A with 250 mM imidazole. Store at 4 oC.

MEP Equilibration Buffer: 20 mM  Phosphate Buffer, pH 7.4, 1M NaCl, 50 mM Arginine. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

MEP Washing Buffer: 20 mM Phosphate Buffer, pH 6.0, 50 mM Arginine. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

MEP Elution Buffer: <strike>20 mM Phosphate Buffer, pH 6.0</strike>, 20 mM NaOAc, pH5.0, 50 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

SP Equilibration Buffer: 20 mM Phosphate Buffer, pH 7.4, 50 mM NaCl, 50 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

SP Washing Buffer: 20 mM Phosphate Buffer, pH 7.4, 500 mM NaCl, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

SP Washing Buffer II: '''200 mM''' Tris-HCl, pH 7.0~pH 7.4, 500 mM NaCl, 0.1 mM EDTA, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

SP Elution Buffer: 20 mM Phosphate Buffer, pH 7.4, 1 M NaCl,  0.1 mM EDTA, 250 mM Arginine, Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.

 Dialysis Buffer I: 500 mM NaCl, 20 mM  Phosphate Buffer, pH 7.4, 50 mM Arginine. Dialysis Buffer II: 50% glycerol, 500 mM NaCl, 20 mM Phosphate Buffer,  pH 7.4, 50 mM Arginine.

1 M Arginine:  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Arginine is very expensive.

'''Expression and Purification'''

 1. Transform <strike>TUNER (DE3)pLacI (Novagen)</strike> Nico21 (DE3) with the Cre expression plasmid <strike>pTriEx-HTNC</strike>, pT7A3-HTNCre

<strike>pET28bCre: C43(DE3) pLysS or Nico(DE3)/pLysN</strike>

<strike>2. Make 7 L '''TB medium''' (1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4 and 0.4% '''glycerol)''', '''or LB'''</strike>

3. Inoculate 200 ml LB medium supplemented with <strike>1% (w/v) glucose</strike>, 50 ug/ml <strike>'''carbenicillin''', </strike>Kanamycin and 34 ug/ml chloramphenicol with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

'''pET28bCre: Kan/Chl''' for Nico(DE3)/pLysN

4. Inoculate LB medium supplemented with 1% (w/v) '''glucose''', <strike>100 ug/ml '''ampicillin'''</strike>, 50 ug/ml Kanamycin and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

'''pET28bCre: Kan/Chl''', 1% (w/v) glucose

5. Induce expression by adding 1 M IPTG solution to a final concentration of 0.1 mM IPTG <strike>0.5 mM (add 0.5 ml of 1 M IPTG to 1 L</strike>) and continue incubation for <strike>1 to 2 hours</strike>

<strike>2 hours</strike>. <strike>or 30 oC for 6 hours</strike>. 25 oC o/n. OD600 should be 2.3 after 2 hours at 37 oC for pET28bCre in C43(DE3) pLysS  cells

For '''Nico(DE3)/pLysN''': induce with 0.1 mM IPTG o/n at 25 oC, or 6 hour at 30 oC.

 6. Pellet cells by centrifugation (6000 rpm, 4 min) and freeze the bacterial pellets at −20 oC, or -80 oC, or dry ice. The cell pellets can be stored at −20 oC for several days, but it is advisable to use them as soon as possible.

7. Thaw pellets in warm water and resuspend in  '''20 to 30 ml 1X Buffer A per liter culture '''using a magnetic plastic stick.

'''pTriEx-HTNC/TUNER (DE3)pLacI:''' resuspend cells in 10 ml of 1X Buffer A (without 500 mM NaCl) per liter culture and then add lysozyme stock solution (80 mg/ml) to a final concentration of 2 mg/ml and incubate at room temperature with continuous stirring for 20 min.

 9. Add equal volume of ice-cold '''TT buffer''' to lysed bacteria, mix well.

10. Homogenize for 1 to 2 min, make sure not a lot of bubbles are genenated. If the mixture is still sticky, add more lysis buffer and TT buffer if it is still sticky, optioally add 25 U/ml benzonase or DNase I to the suspension and incubate for 30 min at room temperature. Sonicate the suspension on ice for 2 min (power 50%, cycles 5).

 11. Centrifuge the lysate for 30 min at 4 oC and  12000 rpm (do not run > 12000 rpm).

Spin again at 30000 rpm for 25 min at 4 oC on ultracentrifuge. Turn off centrifuge for 1 hour if vaccum can not go down, then turn on again.

 12. Decant the supernatant, load to 2.5 cm column packed with 5 ~ 10 ml Ni beads.

Alternatively, the following steps can be used

*add 2 ml 50% Ni-NTA agarose per liter of expression culture (total 14 ml beads, or 28 ml 50% beads), mix well, and pour suspension into 50-ml tubes.

*Incubate the suspension with continuous rotation at 4 oC for 1 h.
*Spin down beads (1 min at 3000 rpm, 4 oC).
*Resuspend beads in lysis buffer and pour the suspension into Econo-Pac® columns (Bio-Rad) or Sigma glass columns and allow the liquid to flow through completely. 

 13. Wash the Ni beads twice with 5 to 20 column volumes of NI Washing Buffer (10 mM imidazole) until no protein is detectable.

14. Equilibrate 5 (or 10) ml MEP column with MEP Equilibration Buffer.

15. Elution of the protein directly to the MEP column with 10 ~15 CV  MEP Elution Buffer,

16. Wash column with at least 10 to 15  CV of MEP Washing Buffer  until no protein is detectable.

17. Elute Cre protein directly to 5 (or 10  ml) of SP or CM column with 10 to 15 CV of MEP Elution Buffer. Close the column valve.

Note: <strike>add 1 vol of dilution buffer (20 mM Phosphate Buffer, pH 7.5) to eluate if elution to CM column.</strike> For SP column dilution is not needed.

After elution, turn on the column valve and adjust flow rate to ~ 1 ml/min so that the Cre protein can bind to SP <strike>or CM resin.</strike>

20. Wash SP <strike>or CM</strike> column with 4 CV of  <strike>CM Washing Buffer or</strike> SP Washing Buffer.

21. Elute Cre from SP <strike>or CM column</strike> with 5 to 10 CV of  <strike>CM Elution Buffer or</strike>SP Elution Buffer, collect 3 ml fractions. Add elution buffer and let the resin to soak in the elution buffer for 1 min before openning the valve. usually 10 fractions are enough.

22. Measure protein concentration in each fraction using a spectrophotomer. Combine fractions containing Cre.

23. After step 25 clean MEP column with 50 mM NaAc, pH 3.5, then wash beads with 10 CV of water then soak resin in 20% Ethanol (for long term storage) or TBS (pH 8.0) with 150 mM NaCl. MEP resin should be cleaned with 0.1 N NaOH after 3 ~ 5 uses.

24. After step 25 clean <strike>CM or</strike> SP column with 2 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water then soak resin in 20% ethanol.

25. Determine protein concentration (OD280); it should be between 60 and 120 uM (approximately 2.5–5 mg/ml).

26. Wash a dialysis tube thoroughly with double-distilled water.  Check if the dialysis tube is leaking.

27. Transfer Cre protein solution to the dialysis tube. Adjust to 1 mM DTT concentration from 2 M DTT (in a box labeled as AMP-DTT, top cube of 4 oC fridge). For example, add 5 ul of 2 M DTT to 10 ml protein solution. Dialyse protein in 70 ml of '''Dialysis Buffer I  '''(in 100 ml beaker) for 3~4 hour in cold room.  Measure OD280. Dialyze Cre protein again for 3~4 h in cold room. Measure OD280.

28. Dialyse with 50 ml of '''Dialysis Buffer II''' in cold room for 4 to 8 hours, mix protein sol with clean pippette every 2 ~ 4 hour. Change buffer then dialyse overnight to concentrate protein, Measure OD280.

29. Wash dialysis tube with 2 X 0.5 ml of 100 mM arginine, combine proteins.

30. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   Sterilize protein by filtration (0.22-um low protein binding syringe filter) inside the tissue culture hood. Aliquot to 1.5 ml tube at ~1.2 mg/vial.   The glycerol stock should have a concentration of 200–450 uM Cre. Add 5, 10, 20, 30, 50 '''ug''' (not ul) protein to each well (in 12 well plate) of 293T cells stably expressing loxP-sfGFP-loxP-TagRFP (clone #1053). Use '''Dialysis Buffer II''' as control to test buffer toxicity.

31. Put vials immediately on ice.  Make sure the protein is '''at least 1 mg per vial''' (give 1.2 mg so customers will not complain). Create a new lot number in the product database, make sure to record all product information including  protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

33. Store the HTNCre vials in a freezer box at −20 oC o/n.

34. Place the freezer box in a foam box, put the foam box in −80 oC freezer. After 12 hours, take the foam box out and store the freezer box in -80 oC freezer. This way the protein can be frozen slowly. If not handle properly the protein will lose enzymatic activity completely.

Cre protein sequence:

MGHHHHHHGM GAAGRKKRRQ RRRPPAGTSV SLKKKRKVSN LLTVHQNLPA LPVDATSDEV RKNLMDMFRD RQAFSEHTWK MLLSVCRSWA AWCKLNNRKW FPAEPEDVRD YLLYLQARGL AVKTIQQHLG QLNMLHRRSG LPRPSDSNAV SLVMRRIRKE NVDAGERAKQ ALAFERTDFD QVRSLMENSD RCQDIRNLAF LGIAYNTLLR IAEIARIRVK DISRTDGGRM LIHIGRTKTL VSTAGVEKAL SLGVTKLVER WISVSGVADD PNNYLFCRVR KNGVAAPSAT SQLSTRALEG IFEATHRLIY GAKDDSGQRY LAWSGHSARV GAARDMARAG VSIPEIMQAG GWTNVNIVMN YIRNLDSETG AMVRLLEDGD

Molecular Weight: 42763 (42. kDa), Extinction Coefficient: 49270. PI=10.30

OD280 X 0.87=mg/ml

Use this link to calculate protein concentration based on OD280:

[http://www.excellgen.net/eln/tools/protein_tools.html http://www.excellgen.net/eln/tools/protein_tools.html]

Use this link to calculate molecular weight and PI

[http://web.expasy.org/cgi-bin/compute_pi/pi_tool http://web.expasy.org/cgi-bin/compute_pi/pi_tool]

=== Cre Recombinase Transduction into ES Cells ===

1. Plate single-cell suspension of mES cells in normal ES medium with 10–15% fetal calf serum.

2. Prepare ES medium containing 10 uM HTNCre protein by diluting an appropriate amount of HTNCre glycerol stock solution with normal ES medium. Sterilize medium with 0.22-um filter.

 3. After 5–6 h or when the cells are attached, wash carefully with PBS.

4. Incubate cells for 8–20 h with HTNCre-containing medium.

For serum-free ES medium, a 5- to 10-fold lower concentration of HTNCre is recommended because FCS inhibits the transduction

5. After protein transduction, wash once with PBS and add normal ES medium.

=== Cre Recombinase Transduction into Undifferentiated hES Cells ===

Culture hES cells in KO/SR medium on tissue culture plates coated with mitotically inactivated mouse embryonic fibroblasts (MEFs). Cells are passaged by manual scraping or collagenase IV treatment.

Note: Cre protein transduction into hES cells is most efficient if the colonies are very small, ideally clumps of few cells. Therefore, the hES cell colonies are dissociated using accutase II (PAA Laboratories) prior transduction. Accutase II has the advantage that it selectively detaches the hES cells from feeder cells if incubation does not exceed 30 min. The single-cell suspension is plated on MEFs again and allowed to adhere for 24 h. Within this time, very small hES cell colonies are formed, which have the optimal size for protein transduction.

Transduction efficiency is optimal for Cre concentrations of 6 uM and transduction times of 6 h. Longer incubation times do not significantly increase transduction efficiency. The optimal cell density for Cre protein transduction is about 50,000 hES cells per cm2 .

1. One day prior to protein transduction plate mitotically inactivated MEF cells on the desired number of tissue culture plates.

2. Remove the medium from the hES cells and add enough prewarmed accutase II solution to cover the colonies evenly. Incubate for 25 min at 37 oC.

3. Detach the cells with KO/SR medium and transfer them to a 50-ml centrifugation tube. Take a sample of 15 l and mix it with the equal volume of trypan blue solution to stain the cells. Take 15 l stained cells, count the cell number in a Fuchs-Rosenthal chamber, and calculate the total number of hES cells. Calculate how many cells are required for the desired number of tissue culture plates.

4. Transfer the calculated volume of the cell suspension to 15- or 50-ml centrifugation tubes and centrifuge for 5 min at 1000 × g and 4 oC.

5. Resuspend the pellet in KO/SR medium and dispense the cell suspension on the MEF cell-coated tissue culture plates. Allow the cells to adhere for 24 h at 37 oC and 5% CO2.

6. Dilute the Cre protein stock solution in KO/SR medium to the desired concentration.

7. Remove the KO/SR medium from the plated hES cells and add enough Cre-containing KO/SR medium to cover the cells evenly.

8. Incubate for 1–6 h at 37 oC and 5% CO2 .

9. Remove the Cre-containing medium, wash the cells once with knockout DMEM and add normal KO/SR medium again.

10. Prior further use of the Cre-transduced cells, allow the cells to recover for at least 24 h.

=== Cre Recombinase Transduction into hES Cell-Derived Neural Precursors ===

Prepare neural precursor cells from hES cells according to a protocol published by Zhang et al. (In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 2001 19, 1129–1133).

Transfer embryoid bodies (EBs) to PO-coated cell culture plates.

Propagate cells for 10 days in ITSFn medium. Within this time period, neural tube-like structures develop in the EB. These structures are isolated mechanically and are propagated as free-floating neurospheres in N2 medium for 2 weeks. Finally, isolate neural precursor cells by enzymatic dissociation of these neurospheres, plate on PO/laminin-coated culture plates and further cultivate in the presence of FGF2 according to Zhang's protocol. Alternatively, human neural precursors may be derived from fetal Central Nervous System (CNS) tissue based on Brüstle's protocol (1998, Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat. Biotechnol. 16,1040–1044). Optimal parameters for Cre transduction in ES cell-derived neural precursors are a Cre concentration of 1 uM and a transduction time of 6 h. For maximum survival, it is crucial that the cells are confluent before transduction.

1. Dilute the Cre protein stock solution in the medium used for the cultivation of neural precursors to a final concentration of 1 uM.

2. Remove the culture medium from the neural precursor cells and add the Cre protein solution. Incubate for 6 h at 37 oC and 5% CO2.

3. Wash the cells twice with DMEM : F12 medium, then add again the suitable culture medium.

4. Before further use, allow the cells to recover for at least 24 h.

== '''Purification of Cre using MEP  column''' ==

1. After step 17, load supernatant to XK 16/20 Column packed with 10 ml Ni resin  using a peristaltic pump.

2. Wash column using 200 ml washing buffer containing 10 mM Imidazol and 500 mM NaCl, 25 mM Tris-HCl, pH8.

3. Elute protein from Ni column directly to 10 ml MEP column using 50 ml buffer B containing 500 mM NaCl, 250 mM Imidazol, 25 mM Tris-HCl, pH8.

4. Wash column with 100 ml TBS, pH 8, containing 200 mM NaCl.

5. Elute Cre with 50 mM NaOAc buffer, pH5.5, containing 50 mM Arginine, 600 mM NaCl.

2 g NaoAc, 116 ul HoAc, 10 g Arginine, 125 ml 4 M NaCl, H2O to 1L

6. Dialyse eluted protein vs 75 ml of dialysis buffer

50% Glycerol 600 mM NaCl

50 mM Arginine 20 mM HEPES pH to 7.4

Change buffer  every 4 hours (2 times), then dialyse o/n.

7. Measure protein concentration using Bradford assay or OD280.

8. Add 5, 10, 15, 20 ul into loxP-GFP-loxP-RFP 293 cell line to do activity assay.

9. Aliquote, label  tubes and store at -20 oC o/n

10. Store vials at -80 oC.

== '''Protocol 2''', ==

Based on [http://www.jove.com/video/1627/engineering-cell-permeable-protein http://www.jove.com/video/1627/engineering-cell-permeable-protein]

1. Inoculate LB/0.5% glucose [g/v]/50 μg/mL carbenicillin with HTNCre glycerol stock using a pipette tip.

2. Grow bacteria at 37°C O/N (for 16 hours).

3. Next day use the densely grown over-night culture to inoculate TB medium with 0.5% glucose, 100 μg/ml Amp (prewarmed to 37 oC) at a ratio of 1:40 or 1:50.

4. Grow bacteria in an incubator at 37°C to OD600 of 1.5.

5. Add 0.5 mM IPTG to induce for 1 h.

6. Spin down bacteria at 5000 rpm for 10 minutes.

7. Store bacteria at -20 oC.

8. Resuspend frozen bacteria pellets in 10 mL lysis buffer per liter flask culture for 15 minutes at room temperature.

9. Add 1 mg/mL lysozyme, incubate at room temperature for 15 minutes while mixing.

10. Add benzonase to 25 U/mL (1:1000) and incubated while mixing for 15 minutes at room temperature.

11. Sonicate on ice for 1.5 min with 0.5 s pulses at 45% of the power.

12. Add 1 mL cold tartaric salt buffer (TSB) per mL suspension while mixing and incubate for 5 min on ice.

13. Take 10 ul lysate for SDS-PAGE analysis (L).

14. Spin at 4°C for 30 min at 12000 RPM. Take 10 ul of supernatant (S) for SDS-PAGE analysis.

15. Transfer supernatnant to fresh 50 mL falcon tubes, add 2 mL of 50% Ni-NTA slurry per liter of initial expression culture, mix gently for 1 h at 4°C in cold room.

16. Pour the suspension into a gravity flow EconoPac column.

17. Take 10 ul flow through for SDS-PAGE analysis (FT).

18. Wash beads twice with 5 bed-volumes of washing buffer. Take 10 ul from washing fractions for SDS-PAGE analysis (W1, W2).

19. Elute HTNCre with 3 bed-volumes of elution buffer, take 10 ul of eluate fraction (E) for SDS-PAGE analysis.

20. Dialyse against High Salt Buffer twice (volume of dialysis buffer to sample is 50), first 1 hour, then O/N at 4 oC.

21. Dialyse against Gylcerol Buffer twice, first 3 ~ 4 hours, then O/N at 4 oC. This will result increase of Cre concentration to 4 folds

22. After dialysis HTCre concentration will be netween 200 and 450 μM, final yield will be ~12 mg per liter of expression culture.

24. Take 1 ul of glycerol stock (GS) for SDS-PAGE analysis.

25. Store HTNCre stock solution at -20°C or -80°C

{| border="1"
|-
! Material Name
! Type
! Company
! Catalogue Number
! Comment
|-
| TUNER (DE3) pLacI
| 
| Novagen
| 70625
| 
|-
| Glycerol
| 
| Carl Roth
| 3783.2
| 
|-
| Na2HPO4
| 
| Roth
| T876.1
| 
|-
| Trizma Base
| 
| Sigma-Aldrich
| T1503
| 
|-
| HCl
| 
| Roth
| 4625.1
| 
|-
| Imidazol
| 
| Roth
| X998.4
| 
|-
| NaCl
| 
| Roth
| 9265.2
| 
|-
| Yeast Extract
| 
| Roth
| 2363.4
| 
|-
| Trypton/Pepton
| 
| Roth
| 8952.4
| 
|-
| K2HPO4
| 
| Roth
| P749.2
| 
|-
| KH2PO4
| 
| Roth
| 3904.1
| 
|-
| Ampicillin
| 
| Sigma
| A9518
| 
|-
| Carbenicillin
| 
| Sigma
| 6344.2
| 
|-
| HEPES
| 
| Sigma
| H3375
| 
|-
| Lysozyme
| 
| Sigma
| 62971
| 
|-
| Benzonase
| 
| Novagen
| 
| 
|-
| L-Tartaric acid, disodium salt
| 
| Sigma
| 
| 
|-
| 50% Ni-NTA slurry
| 
| Invitrogen
| R901-15
| 
|-
| EconoPac columns
| 
| Biorad
| 732-1010
| 
|-
| Sterile filter 0,22μm
| 
| Whatman
| 
| 
|-
| Paraformaldehyde (PFA)
| 
| Sigma
| 
| 
|-
| LB medium
| 
| 
| 
| Yeast extract, Trypton/Pepton, NaCl
|-
| TB medium
| 
| 
| 
|
Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4

|-
| Lysis Buffer (PTB)
| 
| 
| 
| 50 mM Na2HPO4, 5 mM Tris, pH 7.8
|-
| Tartaric Salt Buffer (TSB)
| 
| 
| 
| PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol
|-
| Washing Buffer
| 
| 
| 
| PTB, 500 mM NaCl, 15 mM Imidazol
|-
| Elution Buffer
| 
| 
| 
| PTB, 500 mM NaCl, 250 mM Imidazol
|-
| High Salt Buffer
| 
| 
| 
| 600 mM NaCl, 20 mM HEPES, pH 7.4
|-
| Gylcerol Buffer
| 
| 
| 
| 50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4
|-
| TrypLE™ Express
| 
| Invitrogen
| 
| 
|-
| ESGRO (LIF)
| 
| Millipore
| 
| 
|-
| NEAA
| 
| Gibco
| 11140035
| 
|-
| L-Glutamin
| 
| Gibco
| 25030024
| 
|-
| β-Mercaptoethanol
| 
| Gibco
| 31350010
| 
|-
| DMEM
| 
| Gibco
| 11960044
| 
|-
| PBS
| 
| Gibco
| 
| 
|-
| Fetal Calf Serum (FCS)
| 
| PAA
| 
| 
|-
| X-Gal staining solution:
| 
| 
| 
| 4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS
|-
| K3(FeIII(CN)6)
| 
| Sigma
| P-3367
| 
|-
| K4 (FeII(CN)6)
| 
| Sigma
| P-9387
| 
|-
| MgCl2
| 
| Sigma
| M8266
| 
|-
| X-Gal
| 
| Sigma
| B4252
| 
|}

 '''TB medium 900 ml'''

Tryptone 12 g Yeast extract 24g Glycerol 4 ml Deionized water or MiliQ water to 900ml

Mix well and autoclave for 20 min at 15 psi on liquid cycle, Store at room temp

'''Potassium salt 1000 ml'''

0.17M KH2PO4 and 0.72 M K2HPO4

KH2PO4 23.1g K2HPO4 125.4g Deionized water 900 ml

After the salt has completely dissolved, adjust volume to 1000 ml with D.I. water Sterilize by autoclaving for 20 min at 15 psi on liquid cycle Store at room temp; do not add to TB broth until needed.

'''Complete TB medium''': add 100 ml Potassium salt to 900 ml TB medium.

'''Protein transduction into murine embryonic stem (ES) cells'''

1. Seed ES cells carrying a conditional β-Galactosidase reporter construct  as single cells using TrypLE™ Express for dissociation of adherent cells.

2. After 4 to 6 hours the cells had re-attached and remove the medium. (80% confluency)

3. Incubate ES cells with HTNCre-containing medium for 16 hours.

Dilute an appropriate amount of HTNCre protein (corresponding to 10 μM) from the glycerol stock with ES medium and then sterile filtrated (0.22 μm).

4. After protein transduction change medium back to normal growth medium. 5. After two days wash cells with PBS and fixed with 4% Paraformaldehyde (PFA) for 10 minutes. 6. Wash 2 times with PBS

7. Stain fixed cells with X-Gal staining staining solution and incubate over night at 37°

Important: The product we are selling online is Cre from pTriEx-HTNC (His-Tat-NLS-Cre, clone #298) or pET28b-Cre (clone #375). Both of them produce His-Tat-NLS-Cre.

 If protein expression level is low, use plasmid to transform Rosetta BL21 (DE3) pLysS completent cells and grow bacterial on plate containing Chl/Car (for pTriEX-HTNC) or Chl/Kan (for pET28b-Cre). Pick a few colonies and grow in 5 ml medium containing Chl/Car (for pTriEX-HTNC) or Chl/Kan for pET28b-Cre), make a glycerol stock, use the rest of cells for protein expression.

== Protocol 3 Auto-Induction ==

1. Day 1: Early Morning: innoculate pTriEx-HTNC (His-Tat-NLS-Cre, clone #298),  pET28b-Cre (His-Tat-NLS-Cre in pET28b  vector, clone # 375),  or pET8XHis-Cre (His-NLS-Cre in pET8XHis, clone # 287) transformed Rosetta BL21 (DE3) pLysS cells (Novagen) O/N in ~ 130 ml '''LB/Car/Chl''' (for pTriEx-HTNC) or '''LB/Kan/Chl '''(for  pET28b-Cre or pET8XHis-Cre).

2.  At 5 PM: add 20 ml culture to 1 L  '''LB/Car/Chl or LB/Kan/Chl autoinduce medium. '''Grow 6 L o/n at 30 oC. Note: set temperature to 20 ~25 oC and the incubator will be 30 oC. '''Do not use 37 oC.'''

3. Day 2: In the morning (10 AM to 4 PM, dependent on cell density) : at 6000 rpm for 5 min, wash cells with water and transfer to 50 ml VWR tubes, spin, decant water, add lysis buffer, freezee with dry ice (can store the cell pellet in -80 oC freezer after this step).

'''IPTG-Induction'''

1. Day 1: Late Afternoon: innoculate pTriEx-HTNC (6XHis-Tat-NLS-Cre), or  pET28b-Cre, or pET30-8XHis-Cre (His-NLS-Cre) transformed  Rosetta BL21 (DE3) pLysS cells (Novagen) O/N in ~ 250 ml LB-2%Glucose/Car/Chl (for pTriEx-HTNC) or LB-2%Glucose/Kan/Chl/ (for pET28b-Cre or pET30-8XHis-Cre) O/N at 30 oC.

2. Day 2: In the morning, add 40 ml culture to 1 L LB/Car/Chl or LB/Kan/Chl medium. Grow 6 L at 37 oC until OD600 = 0.6~0.8.

3. Add 0.5 mM IPTG (final conc), induce for 3 ~ 4 hours 30 oC. '''Important: Do not use 37 oC.'''

4. Collect cells by centrifugation at 6000 rpm for 5 min, wash cells with water and transfer to 50 ml VWR tubes, spin, decant water, add lysis buffer, freezee with dry ice (can store the cell pellet in -80 oC freezer after this step). Continue to Purification step 1 and 2.

'''Purification'''

 1. Resuspend cell pellets in 30 ml TB per liter flask culture (6L: at least 180 ml), freeze-thaw with dry-ice then 30 oC water for 1 or 2 cycles (when the cells become glue like gel when the cells are lysed)

2. Use homogenizer to break lysed cells (gel solution).

3. Add 20 ul DNase I and let the bottle rock in cold room overnight.

4. Add 3 ml '''cold''' '''TSB''' to 30 ml suspension.

5. Incubate for 5 min on ice.

6. Spin for 30 min at 12,000 RPM, at 4 oC, Transfer supernatant to clean ultracentrifuge tubes, spin at 30000 rpm at 4 oC for 30 min using ultracentrifuge.

7. Add 10 ml regenerated Ni-NTA beads. Mix by rocking gently in cold room for 60  min to 2 hours.

8. Spin down beads at 6000 rmp for 1 min.

9. Pour supernatant into a clean tube. Be careful not to lose any beads.

10. Pour beads suspension into a gravity flow column, collect flow through, combine flow through and supernatant from step 9.

11. Wash with 5 X 5 bead volumes washing buffer '''or until no protein is detectable in 10 µl eluate + 75 µl Bradford reagent'''.

12. Wash beads with washing buffer (50 mM imidazole).

13. Elute HTNCre or His-NLS-Cre with 3-bed volumes (30 ml) of '''Elution Buffer'''.

14. Dialyse against 75 ml volumes '''dialysis buffer or buffer C''' for 2 hrs. Change buffer every two hours. Repeat 3~4 times.

15. Dialzye against 75 ml '''glycerol buffer or buffer D''' to concentrate protein 2 hour each. Repeat 2 to 3 times.

16. Dialzye against 75 ml '''glycerol buffer or buffer D''' to concentrate protein o/n.

Concentrate protein by dialyzing against 3 X 75 ml Buffer D ---> 450 uM (corresponds to 19 mg/ml) Cre, change buffer every two hours for the first two dialysis, then overnight for the last dialysis.

17. Transfer dialysed liquid to clean ultracentrifuge tube, spin at 30,000 rpm at 4 oC for 20 min.

18. Transfer supernatant to a new 50 ml tube, discard pellet which contains denatured (misfold or inactive) Cre.

19. Make 1:10 serial dilutions of 5 µl protein using Buffer D (not water nor PBS) and measure protein concentration using spectrophotometer and Bradford.

20. Filter through 0.2 µ filter and aliquot into autoclave tubes (1 mg/ml) in the biosafety hood. Avoid forming bubbles.

21. Save a small aliquot for SDS-PAGE analysis to confirm size and purity.

22. Put label (make sure concentration, lot number and size are correct). Upload QC data to ELN. 

23. Put vials in a 96 well paper box,  put the paper box into a styrofoam box (so the protein will be freezed slowing). Store the box in -20 oC o/n, then -80 oC o/N, take out the 96 well box from styroform box and store it at -80 oC.

24. Regenerate Ni beads, add beads to the mixed solution of supernatant from step 9 and flow through from step 10.

25. Repeat step 11 to 20 to purified additional Cre.

This procedure results in glycerol stock solutions containing usually between 200 and 450 micromolar (4~19 mg/ml) HTNCre, '''at least''' 2 mg/ml and total 30 mg HTNCre per column purification should be obtained.Usually, 10 to 50 mg of active Cre can be purified from 1 L culture.

'''Dialysis buffer''':

600 mM NaCl 20 mM HEPES pH to 7.4

'''Glycerol buffer''':

50% Glycerol 600 mM NaCl 20 mM HEPES pH to 7.4

'''TB:'''  Buffer A for PKA alpha purification, i.e., 20 mM Tris-HCl pH 8.0, 600 mM NaCl, 10 mM Imidozale, 10% Glycerol (optional), 0.3 mM EDTA, 2 mM beta-ME. '''TSB:''' tartaric salt buffer, PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM imdazole, Sigma. '''Buffer A''': PTB, 500 mM NaCl, 15 mM imidazole.

'''Washing Buffer: '''TB + 20% glycerol + 20 mM imidazole 

'''Elution Buffer: '''10% glycerol,  250 mM imidazole, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4. '''Buffer B''': PTB, 500 mM NaCl, 250 mM imidazole. '''Buffer C:''' 600 mM NaCl, 20 mM Tris-HCl, pH 7.4, 20% glycerol. '''Buffer D''': 50% glycerol, 500 mM NaCl, 20 mM  Tris-HCl, pH 7.4

or 1 X PBS, with additional 250 mM NaCl and 50% glycerol.

'''Cre Recombination Assay (0.5 to 10 uM Cre is sufficient)'''

1. Culture CV1-5B and COS-7 cells on a 10 cm2 dish culture plate in normal culture medium for 24 h. 2. Dilute HTNCre glycerol stock solution with culture medium. 3. Filter sterilization with a 0.22 microM filter. 4. Culture cells for indicated time periods in medium containing purified recombinant HTNCre in in a 1:1 mixture of PBS and Glutamax supplemented DMEM (Invitrogen). * For long-term incubations (over 16 h) , dilute HTNCre to 5 uM with normal FCS-containing medium. * For growth curve analysis, wash cells were once with PBS after transduction and further cultivate for 5 h in normal medium before being trypsinized and passaged. * For LacZ reporter gene assays, wash cells after transduction and culture for further 24 h in normal medium before fixation. * For transduction into ES cells, plate single suspensions of cells in culture medium 6 h prior to Cre transduction, then culture cells for 18 h with recombinant HTNCre in normal ES cell medium. After transduction, wash cells once with PBS and further cultivated for 2 days in normal medium before being further passaged. * For visualization of HTNCre translocation, incubate CV1-5B cells 30 min with R-HTNCre and wash three times with PBS (Gibco) containing 0.5 mg/ml heparin (Sigma), 5 min each, to remove extracellular bound protein. Use Hoechst 33342 (1 uM) for nuclear counterstaining.

== Protocol 4 for high concentration Cre ==

Immediately after eluting Cre out from column, use Desalting column to desalt (15 ml per injection). Collect and combine enzyme fractions, transfer to 50 ml tubes,  add Ni beads, incubate in cold-room using rotation mixer for 30 min, spin down (1000 rpm, 1 min), decant liquid (do not discard), combine beads, wash with Dialysis buffer, spin, remove as much liquid as possible, add minimal vol of Elution Buffer, soak for 15 min, spin gentle, use pippet to transfer liquid to dialyse tube. Dialyse as step 14 and forward in protocol 1

== Protocol 5 ==

Large scale (6L) purification of TAT-Cre (by Jing Wang)

 Competent cells: E.coli Tuner (DE3) pLacI (Novagen) Plasmid: pTriEx (Novagen)-His-TAT-NLS-Cre

PLacI (Novagen) contaning pTriEx -His-TAT-NLS-Cre (Novagen)

1. Inoculate 150ml LB medium. (50ug/mg Carbenicillin---sigma and 34 ug/ml chloramphenicol---sigma in 500ml flask and grow O/N at 37 oC.

 2. Remove 1ml LB for OD600 blank. Add 40ml culture to 1L pre-warmed LB (50ug/ml Carbenicillin and 34ug/ml Chloramphenicol) in 2L flasks. Grow at 37C until OD600 is <0.600.

 3. Aliquot 100 ml for control.

 4. Add 0.5 mM IPTG (2ml, 0.5M stock to each 4L flask) and grow for another 3-4 hrs. Check OD again if possible.

 5. Cool down on ice for 10min and harvest in 1L conical flasks and spin 6000 RPM, 4oC for 10 min, aspirate supernatant completely and freeze pellets on dry ice/ethanol both, store at -80C.

'''Bacterial Lysis'''

1. Prepare lysis buffer 100ml, take the frozen bacterial on ice and add 10ml to each 1L flask. Wait a few min on ice and resuspend well bacterial pellets, wash with 5ml the same buffer. Transfer to new tubes. Gently mix and incubate 15 min at RT.

2. Add Bugbuster detergent (Novagen#70921), gently mix for 15min RT.

3. Transfer to centrifuge tubes and spin down at 16,000rpm, 4C, 25min.

4. Pool all cleared lysate and filter through 0.22um. Save 100ul for SDS-PAGE analysis.

 '''Nickel Column'''

1. Wash Ni-NTA slurry (Qiagen) twice with bead wash buffer (prep. 100ml). resuspend in 1 Vol. bead wash buffer.

2. Save 4ml slurry for pre-loading (2ml/ea.).

3. Divide left-over slurry to 5---50ml tubes. Spin (500rpm, 1min) and aspirate bead wash buffer.

4. Calculate cleared lysate evenly. 1ml packed bead/ 6ml cleared lysate and add to the slurry.

5. Mix gently shaking at 4C at least 1-2hr.

6. Load onto 2 plastic columns (Bio-rad Econo-columns, cat.#737-1512). Preload with the saved 2m l each bead slurry.

7. Let lysate flow through. Save 100ul for SDS-PAGE.

8. Wash column with >10 vol. column wash buffer, O/N Is OK

9. Elute column with elution buffer (100ml), collect 2ml fraction each in cold room.

10. Determine pro-con. By Bradford Assay and pool fractions.

'''Dialysis'''

1. Dialyze (10K MWCO, Slidelizer, Perirce) against 100 vol. dialysis buffer >2hrs, repeat 1X or O/N.

 2. Dialyze against 4L glycerol buffer to concentrate. Filter through 0.22 mm filter.

Aliquot in hood and slow freeze (-20 C O/N, transfer to -80C).

 '''Clean column'''

1. wash w/ 2 vol. 6M GuHCl, 0.2 M acetic acid. 2. wash w/ 10 vol dH2O. 3. wash w/ 3 vol 2% SDS. 4. wash w/ 1 vol. 25% EtOH. 5. wash w/ 1 vol. 50% EtOH. 6. wash w/ 1 vol. 75% EtOH. 7. wash w/ 1 vol. 100% EtOH. 8. wash w/ 1 vol. 75% EtOH. 9. wash w/ 1 vol. 50% EtOH. 10. wash w/ 1 vol. 25% EtOH. 11. wash w/ 5 vol. dH2O. 12. wash w/ 5 vol. 0.1MEDTA, pH8.0 13. wash w/ 10 vol dH2O. 14. recharge with 5 vol. 0.1M NiSO4. 15. wash w/ 10 vol dH2O. transfer to tubes, 4C store.

'''Solutions:'''

 '''Stock 1''' 10X phosphate buffer: 0.5 M NaHPO4 70.89g 50mM Tris 6.06g adjust pH to 7.8, filter 2L

'''Stock 2 ''' 5M NaCl

'''Stock 3 ''' 1M imidazole adjust pH 7.0, filter

'''Lysis buffer''' 100ml '''10X phosphate buffer''' 10ml (stock1) 0.5M NaCl 10ml (stock2) 10 mM imidazole 1ml 1X protease inhibitors (EDTA-free tablets, Roche) 10 tablets 2 mM PMSF 1mg lysozyme/ml 100mg Benzonase (1:500) 79ml dH2O

Bead wash buffer column wash buffer column elution buffer (100ml) (1L) (100ml) Stock1 10ml 100ml 10ml Stock2 10ml 100ml 10ml Stock3 1.5ml 20ml 25ml dH2O 78.5ml 780ml 55ml

phosphate 1x buffer NaCl 0.5M 0.5M 0.5M Imidazole 15mM 20mM 250mM

 '''Dialysis buffer:''' 0.6M NaCl 20 mM HEPES pH 7.4

'''Glycerol buffer: ''' 50% Glycerol 0.6M NaCl 20 mM HEPES pH 7.4 Notes: 1. keep cold as normal protein purification. 2. wear gloves, TAT-Cre can transducer your cells too.

== Protocol 6 ==

Large Scale Purification of TAT-Cre (HTNC) Under Native Conditions Developed by: Michael Peitz(m.peitz@uni-koeln.de), Frank Edenhofer(peitz@uni-bonn.de) Slightly Modified by: Kevin Otipoby (kotipoby@cbr.med.harvard.edu)

'''Expression in bacteria ''' TUNER(DE3)pLacI (Novagen) containing pTriEx(Novagen)-His-TAT-NLS-Cre 1. Inoculate 150 ml LB medium containing 50 ug/m1 Carbenicilli and 34 ug/m1 Chloramphenicol in 500 ml flask and grow overnight at 37°C with shaking.

2. Add 120 ml overnight culture to 6 liters prewarmed LB containing 10011g/m1 Ampicillin (or 50ug/m1 Carbenicillin) and 34ug/m1 Chloramphenicol in three 4 liter flasks.

3. Grow at 37°C with shaking until OD 600 is 0.9-1.0 (approximately 1.5 - 3 hours).

4. Induce expression with 0.5 mM IPTG.

5. Grow cultures for an additional 3 - 4 hours.

6. Cool down flasks quickly in ice bath for 10 min and harvest cells in 6 orange-capped conical flasks (250 ml) by centrifuging at 4200 RPM (Sorvall), 4°C, 20 min. This requires 4 spins; pour off supernatant from each tube and add an additional 250 ml on top of pellet. Once the entire 6 liters have been spun down, aspirate supernatant completely and freeze pellets on dry ice/ethanol bath; store at -80°C.

'''Bacterial Lysis''' 7. Thaw and resuspend bacterial pellets in 3 ml resuspension buffer / gram wet weight of bacteria until completely resuspended. Add 1 mg lysozyme/ml suspension.

8. Mix, incubate 20 min at RT.

9. Add 1:1000 Benzonase for additional 15 min at RT.

10. Sonicate 2 min with 0.5 sec pulses on ice.

11. Add 1 ml tartaric salt buffer/ml suspension.

12. Mix gently and incubate for 5 min on ice.

13. Transfer to centrifuge tubes and spin at 16,000 rpm (SS34, Sorvall), 25 min, 4°C.

14. Pool cleared supernatant. Filter through 0.22 um filter (this step takes a while and probably requires multiple changes of filters).

'''Nickel Column Purification'''

15. Wash Ni-NTA slurry (Qiagen) twice with bead wash buffer. Resuspend packed beads in 1 volume bead wash buffer.

16. Add 2 ml bead slurry (1 ml packed beads) / 6 ml cleared lysate and mix by gently shaking at 4°C for 1 hr.

17. Load onto 2 plastic columns (Bio-Rad Econo-columns, cat. #737-1512) preloaded with 2 ml bead slurry (1 ml packed beads).

18. Let lysate flow through.

19. Wash with at least 10 bed volumes wash buffer.

20. Elute with elution buffer collecting 2 ml fractions.

21. Determine which fractions contain protein by Bradford Assay and pool protein-containing fractions. If solution gets turbid, add more elution buffer until protein is dissolved.

22. Determine protein concentration by Bradford Assay with BSA standard and (optional) endotoxin contamination by LAL Assay.

 '''Dialysis''' 23. Dialyze (10K MWCO, Slidelizer, Peirce) against 100 volumes dialysis buffer 2 hrs (minimum). Repeat 1x.

24. Dialzye against 1 liter glycerol buffer to concentrate.

25. Filter through 0.22 um filter, aliquot in sterile tubes.

26. Freeze (place the vials in a Styrofoam container in the —20° C overnight), then Transfer to —80°C freezer.

'''Solutions''' 10x phosphate buffer: 0.5 M Na2HPO4 50 mM Tris adjust pH to 7.8, sterile filter

imidazole stock

1 M imidazol adjust to pH 7.0 (with conc. HO), sterile filter

resuspension buffer: 1 x phosphate buffer 500 mM NaCl 10 mM imidazole 0.5x protease inhibitors (EDTA-free tablets, Roche)

Lysis enzymes

rlysozyme ( ovagen) Benzona (Novagen)

bead wash buffer 1x phosphate buffer 20mM imidazole 500 mM NaCl

tartaric salt buffer: 2M L-Tartaric acid (Disodium salt) lx phosphate buffer 15 mM imidazole

column wash buffer: 1x phosphate buffer 500 mM NaCl 20 mM imidazole

column elution buffer: 1x phosphate buffer 500 mM NaCl 250 mM imidazole pH to 7.4

glycerol buffer: 50% Glycerol 600 mM NaC1 20 mM HEPES pH to 7.4

Dialysis buffer 600 mM NaC1 20 mM HEPES pH to 7.4

 Basic protocol for TAT-Cre transduction

Harvest organs and purify cells as normal.

Wash cells 3x in Hyclone ADCF-Mab serum-free media. If you do not wash 3x, your efficiencies will be lower (probably about 25-35%). Resuspend cells at 107/m1 in Hyclone serum-free media. Dilute TAT-Cre to 100 pz/ml in Hyclone serum-free media. Prewarm to 37 degrees C for about 10 min. Add 1:1 cells and TAT-Cre (or Hyclone media for a —TAT-Cre control). Final conc of cells = 5x106 cells/ml Final cone of TAT-Cre = 50 1.1g/m1 (1.25 11M) Incubate 45 min at 37 degrees C. Stop transduction with media containing 10% FCS. If the volume of cells and TAT-Cre is large, I add FCS to final conc of 10%, then fill the tube with media containing 10% FCS. Spin, wash cells with media containing 10% FCS. Place in culture.

Notes: 1) Wear gloves when handling TAT-Cre; it will transduce your skin cells as well as lymphocytes. 2) Transduction efficiency should be around 45-55% in primary B cells. 3) There is some toxicity associated with TAT-Cre transduction. You should recover at least 75-80% of the cells. If you go up on the dose of TAT-Cre or longer on the time of incubation, the transduction efficiency does not go up but the recovery goes down. This might not be the case for cells other than primary B cells, though. 3) Transduction in the presence of synthetic TAT-HA (see Wadia et at., Nature Medicine. 2004) allows one to use TAT-Cre at lower doses which gives near 100% recovery of cells. 4) You can transduce cells in the presence of FCS, but you have to use much more TAT-Cre.

5), opti-MEM can be used to replace Hyclone ADCF-Mab serum-free media

Reference

for construction of HTNC plasmid: Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F. 2002 PNAS 99(7):4489-94. Other relevant references for TAT-Cre:

Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE. 2001 Nature Biotechnology19(10):929-33. Joshi SK, Hashimoto K, Koni PA. 2002 Genesis 33(1):48-54. Wadia JS, Stan RV. Dowdy SF. 2004 Nature Med 10

== Cre activity assay ==

Cre purified with

*1. Ni column only: High activity, high toxicity
*2. Ni column then SP: medium activity, medium toxicity
*3. Ni column then MEP: very low activity, low toxicity.Cre Expression/Induction using Auto-Induce, IPTG and L-arabinose

Clone 1708, and 1010

[[File:Cre expression.jpg|RTENOTITLE]]

Load Cre from 1010 to 20 ml QFF-->50 SP-FF, elute with 300 ml 0f 100 mM-->1M NaCl linear gradient 

[[File:Cre 1010 Purification SPFF.jpg|RTENOTITLE]]

Expression screening using 10 ul magnetic beads from expedeon Amintra® Nickel Magnetic Affinity Resin

[[File:Cre375 2603 2642 2643 2644.jpg|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| ID
| Name 
| Host
| MW
| Induction
| Note
| Expression
|-
| 1
| 375
| pET28-Cre 
| Nico21(DE3)pLysN, 
| 10.30 / 42.8 
| 1 mM IPTG, 18 oC
| grown on LB/KC, most cells are lysed 
| y
|-
| 2
| 375
| pET28-Cre 
| Rosetta(DE3)
| 10.30 / 42.8 
| 1 mM IPTG 
| looks like contaminated, can not lyse cells
| trace
|-
| 3
| 375
| pET28-Cre
|
Nico21(DE3)pLysN

| 10.30 / 42.8 
| 1 mM IPTG 
| grown on LB/K 
| y
|-
| 4
| 2603
| pL6H-sso7d-SD-Taq 
| Nico21(DE3)pLysN 
| 6.10 / 104.7 
| 1 mM IPTG 
| 
| y
|-
| 5
| 2642
| pET29b-Flp 
| Nico21(DE3)pLysN 
| 9.76 / 52.5 
| 1 mM IPTG 
| most cells are lysed
| n
|-
| M
| 
| 
| pageruler plus
| 
| 
| 
| 
|-
| 6
| 2643
| pET29b-Int-C3 
| Nico21(DE3)pLysN 
| 9.69 / 41.4 
| 1 mM IPTG 
| 
| maybe
|-
| 7
| 2643 
| pET29b-Int-C3 
| Nico21(DE3)pLysN 
| 9.69 / 41.4 
| 1 mM IPTG 
| 
| maybe
|-
| 8
| 2644
| pET29b-scIHF2 
| Nico21(DE3)pLysN 
| 9.60 / 22.8 
| 1 mM IPTG 
| 
|  ?
|-
| 9
| 1010
| Cre
| Nico21(DE3)pLysN 
| 
| 
| Lot 174-1010
| 
|-
| 10
| 1186
| MBP or MBP-Xis 
| 
| 
| 
|
| 
|}

[[File:Cre purification magnetic1.jpg|RTENOTITLE]]

Purification of Cre using Ni-NTA mag beads from Genscript and Ni_NTA agarose from GE

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Sample
| 
| 
| 
|-
| 1
| Ni-NTA Mag beads sample 1 frac2
| 
| 
| 
|-
| 2
| Ni-NTA Mag beads sample 1 frac3 
| 
| 
| 
|-
| 3
| Ni-NTA Mag beads sample 1 frac4 
| 
| 
| 
|-
| 4
| Ni-NTA Mag beads sample 2 frac2 
| 
| 
| 
|-
| 5
| Ni-NTA Mag beads sample 2 frac3 
| 
| 
| 
|-
| 6
| Ni agarose column  16mm    frac 1
| 
| 
| 
|-
| 7
| Ni agarose column  16mm    frac 2 
| 
| 
| 
|-
| 8
| Ni agarose column  2.5 cm  frac 8 
| 
| 
| 
|-
| 9
| Ni agarose column  2.5 cm  frac 11 
| 
| 
| 
|-
| 10
| Ni agarose column  2.5 cm  frac 14 
| 
| 
| 
|-
| 11
| Ni agarose column  2.5 cm  frac 17 
| 
| 
| 
|-
| M
| pageRuler Plus
| 
| 
| 
|}
[[Category:Protocols]]

Cre Production

2022-02-15T23:38:52Z

Paul.xu: Paul.xu moved page Cre Production to Cre QC

#REDIRECT [[Cre QC]]

Cre QC

2022-02-15T23:38:52Z

Paul.xu: Paul.xu moved page Cre Production to Cre QC

[[File:Cre purification 5 18 19.jpg]]
[[Category:QC]]

Cleancap mRNA Invitro Transcription

2022-02-13T16:14:04Z

Paul.xu:

= Protocol for Co-transcriptional capping using CleanCap® Reagent AG from TriLink and HiScribe™ T7 High Yield RNA Synthesis Kit from New England Biolabs® =

'''Reagents required:'''

1. NEB HiScribe™ T7 High Yield RNA Synthesis Kit, NEB #  [https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit] E2040S  2. TriLink CleanCap® Reagent AG, N-7113, or CleanCap® Reagent AG (3’ OMe), N-7413

https://www.trilinkbiotech.com/media/catalog/product/cache/c687aa7517cf01e65c009f6943c2b1e9/n/-/n-7413.png

{|
|-
! scope="row" | Extinction Coefficient
| data-th="Extinction Coefficient" | 30,539 Lmol-1cm-1 at 255 nm
|-
! scope="row" | Molecular Formula
| data-th="Molecular Formula" | C33H45N15O24P4 (free acid)
|-
! scope="row" | Molecular Weight
| data-th="Molecular Weight" | 1159.60
|-
! scope="row" | Salt Form
| data-th="Salt Form" | Na+
|-
! scope="row" | Concentration
| data-th="Concentration" | 100 mM
|-
! scope="row" | Buffer
| data-th="Buffer" | H2O
|-
! scope="row" | Recommended Storage
| data-th="Recommended Storage" | -20°C or below
|-
! scope="row" | Other Name(s)
| data-th="Other Name(s)" | CleanCap®Reagent AG (3' OMe) for co-transcriptional capping of mRNA, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG
|}

10 nmole ==>11.6 mg

to make 100 mM, 11.6 mg + 100 ul H2O

to calculate more acculately, dilute 1000 fold, use nanodrop, 30,539 Lmol-1cm-1 at 255 nm

=== ARCA (m27,3'-OGP3G or 3′-O-Me-m7G(5′) ppp(5′)G ===

[[File:ARCA Cap Analog.png|RTENOTITLE]]

=== CleanCap AG (3'OMe) ===

[[File:CleanCap AG (3'OMe)n-7413.png|RTENOTITLE]]

=== ===

[[File:ARCA Cap Analog.png|RTENOTITLE]]

=== Cap analogues ===

[[File:Cap annalogues.png|RTENOTITLE]]

== ==

== [[File:Cleancap vs ARCA.png|RTENOTITLE]] ==

Pfizer

[[File:Phfizer GA cap.png|RTENOTITLE]]

== Trinucleotide cap1 analogue ==

((m2 7,3’-O)Gppp(m2’-O)ApG; TriLink)

== ==

== UTP  ==

Uridine 5'-Triphosphate, Sigma , 94370-1G, 1 g $288

[[File:Uridine-5'-Triphosphate.png|RTENOTITLE]]

Pseudouridine-5'-Triphosphate, Pseudo-UTP, 5-Ribosyl Uracil,  N-1019-5,5 µmole $451

Pseudouridine (5-ribosyluracil) was the first modified ribonucleoside discovered. It is the most abundant natural modified RNA base, and has been deemed the "fifth nucleoside" in RNA. It can be found in structural RNAs, such as transfer, ribosomal and small nuclear RNA. Pseudouridine has been found to enhance base stacking and translation. Pseudouridine-5'-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors with in vitro transcribed RNA. Pseudo-UTP, along with 5-methylcytidine-5'-triphosphate (5-methyl-CTP) has shown innate immune suppression in culture and in vivo while enhancing translation in recent publications. For example, Warren et al. determined an efficient means of reprogramming multiple human cell types using modified mRNA that can express the four primary reprogramming proteins. These cells are referred to as induced pluripotency stem cells (iPSCs). Warren et al. found that enzymatically synthesized RNA substituted with Pseudo-UTP, 5-Methyl-CTP and ARCA effectively evaded the cell’s innate immune response, a crucial component in their success. Reduced toxicity due to substitution with Pseudo-UTP, 5-Methyl-CTP was critical since it allowed repeated transfection with in vitro transcribed mRNA over several weeks.

[[File:Pseudouridine-5'-Triphosphate.png|400px|RTENOTITLE]]

5-Methyluridine-5'-Triphosphate ,  5-Methyl-UTP, 5-Me-UTP, 5-mUTP, Trilink: N-1024-5,5 µmole $464.

Thermostability Enhancer

[[File:5-Methyluridine-5'-Triphosphate.png|RTENOTITLE]]

N1-Methyl-Pseudouridine-5'-Triphosphate, N1-Methylpseudo-UTP, 1-Methylpseudo-UTP, Trilink: N-1081-5,5 µmole $464

Incorporation of N1-methylpseudouridine can reduce the immunogenicity of the resulting mRNA

[[File:N1-Methyl-Pseudouridine-5'-Triphosphate.png|RTENOTITLE]]

'''Reagents not supplied:'''

RiboMAX™ Large Scale RNA Production Systems – T7: Promega P1300 ScriptCap™ m7G Capping System: CellScript C-SCCE0625 ScriptCap™ 2'-O-Methyltransferase Kit: CellScript C-SCMT0625 A-Plus™ Poly(A) Polymerase Tailing Kit: CellScript C-PAP5104H Antarctic Phosphatase: New England BioLabs M0289S ScriptGuard™ RNase Inhibitor: CellScript C-SRI6310K

Lipofectamine™ MessengerMAX™ Transfection Reagent: ThermoFisher LMRNA001 RiboJuice™ mRNA Transfection Kit: MilliporeSigma TR-1013

== Design of Transgene into T7-VEE ==

1, add Kozak after Nde1

Nde1     Kozak     start

catatg gaa t tcc gcc acc ATG---

2, Add stop codon and Kozak after Nde1

Nde1        stop     Kozak start

catatg agg ttc tag gcc acc ATG
<pre> -17 +1-->
T7 promoter class III 5'- TAA TAC GAC TCA CTA TA G GGA GAC ATG CTA - 3'
T7 promoter class II 5'- TAA TAC GAC TCA CTA TT A GGG AGA ATG CTA - 3'

T7 RNA Polymerase
+ rNTPs
---> RNA 5'- pppGGGAGACAUGCUA

</pre>

== 5'UTR ==

1,  T7 + beta globin + kozak

TAATACGACTCACTATAaGG '''GAATACAAGCTACTTGTTCTTTTTGCA '''AATTGCCACCATG

2, T7 +  Venezuelan equine encephalitis virus 

TAATACGACTCACTATAaGG '''ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA'''

== Template preparation ==

1. Double-stranded DNA template

'''**NOTE: CleanCap AG requires modification to the sequence just downstream of the T7 promoter sequence, replacing the “GG” that follows the T7 promoter sequence with an “AG” as follows:'''

'''Standard T7 promoter (underlined) with ''''''GG'''''':'''

'''5’ TAATACGACTCACTATA''''''GG'''''' 3’'''

'''Sequence required for CleanCap® with AG:'''

'''5’ TAATACGACTCACTATA''''''AG'''''' 3’'''

'''New primer'''

'''T7-mRNA-F ''' (for pCDNA5 plasmid) (Box O1well I3)

AAAT'''TAATACGACTCACTATA'''''AG''

'''T7-mRNA-F2''' (for pCDNA5 pCMV5-frt plasmid)

AT'''TAATACGACTCACTATA'''''AG''GAGACCCAAG

'''T7-mRNA-F3''' (for plasmid with traditional T7 promoter and kozak seq, such as pMiniT2--IRES)

'''TAATACGACTCACTATA'''''AG''GAGG

'''T7-mRNA-F4''' (for plasmid vector pCAGGS, 2724)

'''TAATACGACTCACTATA'''''AGGCAAGGCCACCGCCACCATG''

Reverse primer for 2724 pCAGGS: pCAGGS-mRNA-R2

tTTTTTTTTTTTTTTTTTTTTTTTAAATGATTTGCCCTCC (GGAGGGCAAATCATTTAAAAAAAAAAAAAAAAAAAAAAAa)

reverse primer for C-Flag tag

'''Flag-R''' (CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)  TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG

2. NEB Monarch® RNA Cleanup Kit (500 µg),   [https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug] T2050 

'''For pMiniT2-pA vector ''' pA-T7-F TAATACGACTCACTATAGGGAGAC

pA-mRNA-R TAGGTAATCGTCACCTGcag (ctgCAGGTGACGATTACCTA)

'''Co-transcriptional Capping Protocol:''' ('''NOTE''': '''5 mM final each NTP''' instead of 10 mM in standard HiScribe Reaction)

#Thaw the necessary components, keep the T7 RNA Polymerase Mix on ice. 
#Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes. 
#Set up the reaction at '''room temperature''' in the following order:

{| border="1" cellspacing="0" cellpadding="0"
|-
! width="259" |
'''Component'''

! width="134" |
'''Volume'''

! width="197" |
'''Concentration'''

|-
|
'''Nuclease-free Water'''

|
'''X µl'''

|

|-
|
'''10X Reaction Buffer'''

|
'''2 µl'''

|
'''0.5X final'''

|-
|
'''100 mM ATP'''

|
'''2 µl'''

|
'''5 mM final'''

|-
|
'''100 mM GTP'''

|
'''2 µl'''

|
'''5 mM final'''

|-
|
'''100 mM pseudo-UTP'''

|
'''2 µl'''

|
'''5 mM final'''

|-
|
'''100 mM 5m-CTP '''

|
'''2 µl'''

|
'''5 mM final'''

|-
| Cyanine 5-UTP, optional 
| 0.5 '''µl'''
| 
|-
|
'''100 mM CleanCap''''''®'''''' AG or AG (3’ OMe) (N-7113 or N-7413)'''

|
'''1.6 µl'''

|
'''4 mM final'''

|-
|
'''Linear Template DNA'''

|
'''X µl'''

|
'''1 µg total'''

|-
|
'''T7 RNA Polymerase Mix'''

|
'''2 µl'''

|

|-
|
'''Total'''

|
'''40 µl'''

|

|}

1.Mix thoroughly by pipet and pulse-spin in a microfuge. Incubate at 37°C for 2 hours in a dry air incubator or PCR machine to prevent evaporation.

2. Follow the protocol provided with the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S ) for DNase treatment and RNA purification.

3. Add TURBO DNase (ThermoFisher Scientific, incubated at 37 ◦C for 30 min Purify with column

4, Add 5 µL of 10×Phosphatase Buffer to the reaction. 

5, Add 1 µL of RNase-free DNase  

6,  Add 2 µL Antarctic Phosphatase (New England Biolabs) inccubate 37 ◦C for 30 min ( Phosphatase treatment reduces innate immune response in mammalian cells)

'''5m-CTP'''''' ''': 5-Methylcytidine-5'-Triphosphate

'''pseudo-UTP'''''': '''Pseudouridine-5'-Triphosphate

Cyanine 5-UTP, 100 nmol [https://www.perkinelmer.com/product/cyanine-5-utp-nel583001ea NEL583001EA $646.00 ]  , [https://www.enzolifesciences.com/ENZ-42506/cyanine-5-utp-enhanced/ ENZ-42506, 250 nmol, $354.00 ]  , [https://www.scbt.com/p/cyanine-5-utp sc-499469, 250 nmol $340]

Cyanine 3-UTP, 100 nmol [https://www.perkinelmer.com/product/cyanine-3-utp-nel582001ea NEL582001EA  $641] [https://www.apexbt.com/cy3-utp.html 25ul (10mM) $195 ]  [https://www.scbt.com/p/cyanine-3-utp sc-499471 250 nmol $350]

== '''Large scale production''' ==

CleanCap AG (TriLink N-7113) and RiboMAX™ Large Scale RNA Production Systems-T7 (Promega P1300)

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Component 
| Large Scale
| Mid Scale
| Small scale
|-
| T7 Transcription Buffer 5X 
| 20 μL 
| 
| 
|-
| 100 mM ATP 
| 7.5 μL
| 
| 
|-
| 100 mM GTP 
| 7.5 μL 
| 
| 
|-
| 100 mM 5mCTP 
| 7.5 μL 
| 
| 
|-
| 100 mM pseudo-UTP 
| 7.5 μL 
| 
| 
|-
| CleanCap AG, 100 mM  
| 7.5 μL 
| 
| 
|-
| DNA plasmid (1 μg/μL) 
| 10 μL 
| 
| 
|-
| RNAse-free water
| 22.5  μL
| 
| 
|-
| Enzyme Mix (T7) 
| 10 μL 
| 
| 
|-
| Total
| 100 μL 
| 
| 
|-
| colspan="4" | Incubate at 37°C for 2 hrs. Do not incubate beyond 2 hours at 37°C as a longer incubation period may result in increased RNA degradation. 
|-
| 
| 
| 
| 
|-
| colspan="4" | Add 10 μL RQ1 RNase-free DNase (Promega M6101) for 10 μg of template DNA (use 1 μL per μg DNA template). Gently mix by pipetting up and down. Incubate for 15 minutes at 37˚C. 
|-
| Yield
| 300-500 μg. 
| 
| 
|}

For making PCR fragment for in vitro transcription

'''Method 1:''' making T7 promoter + BGH tail

CMV-seq-F 

GAGGTCTATATAAGCAGAGCTC

BGH-mRNA-R

GGTTCTTTCCGCCTCAGAAG

'''Method 2''': making modified promoter AG + BGH tail

'''T7-mRNA-F1 ''' (for pCDNA5 plasmid) 

AAAT'''TAATACGACTCACTATA'''''AG''

'''or T7-mRNA-F2''' (for GOI in pCDNA5 and pCMV5-Frt plasmid vector)

AT'''TAATACGACTCACTATA'''''AG''GAGACCCAAG

BGH-mRNA-R 

GGTTCTTTCCGCCTCAGAAG 

with Flag Tag and polyA: use '''Flag-R'''

 TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG

(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA) PCR product sequence

AT'''TAATACGACTCACTATA'''''AG''GAGACCCAAG
<pre style="color: rgb(0, 0, 0); font-size: 12px; background-color: rgb(251, 251, 239);">ATTAATACGACTCACTATAAGGAGACCCAAGCTGGCTAGC
GCTAGC CACCATG--GOI --ggatcc gactaCAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA</pre>

'''Method 3:''' make T7 promoter + IRES+ BGH tail

first PCR

fragment 1

 2711-IRES-F

ACGATGATAATATGGCCAGCTAGCCACCATGTTCGTCTTC 

BGH-mRNA-R 

GGTTCTTTCCGCCTCAGAAG 

fragment 2

T7-mRNA-F3 (for plasmid with traditional T7 promoter and kozak seq, such as pMiniT2--IRES )

TAATACGACTCACTATAAGGAGG

IVT-IRES-R

CATGGTGGTGGCCATATTATC

2nd PCR

'''T7-mRNA-F3''' (for plasmid with traditional T7 promoter and kozak seq, such as pMiniT2--IRES)

'''TAATACGACTCACTATA'''''AG''GAGG

TAATACGACTCACTATAAGGAGgccACCATG

GH-mRNA-R 

GGTTCTTTCCGCCTCAGAAG

'''For GOI in pCMV5-Frt vector ''' '''T7-mRNA-F3'''  

'''TAATACGACTCACTATA'''''AG''GAGG

'''Method 4:''' make T7 promoter + polyA

For example: 2683 pJet1.2-SARS2-S1-1 PCR using

T7-mRNA-F3 '''TAATACGACTCACTATAAGGAGG'''

on fragment on pJet1.2

'''TAATACGACTCACTATAAGGAGG''' TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTT

CTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA PCR Template Preparation 1. Cloning of IDT gene fragment into pMiniT2 or pJet1.2 2683 (DS1) TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA

 2. Make miniprep, send miniprep DNA out for sequencing (Genewiz, Macrogen) 3, PCR   1 ul of plasmid (50~100 ng) 1 ul of 10 uM T7-mRNA-F3 TAATACGACTCACTATAAGGAGG 1 ul 1 uM 2683-R2 2683-R2 TTACTTGTCGTCATCGTCTTTGTAGTCACCAGCTGTCCAACCTGAAG (CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAA) 1 ul 10 uM Flag-R  TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG

(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA) 21 ul H2O + 25 ul 2 X Phusion PCR mix (NEB)

PCR: 2 min at 95 oC 30 cycles of 95 oC 30 sec, 55 oC 30 sec, 72 oC 1 min 7 min at 72 oC. Final Product 802 bp TAATACGACTCACTATAAGGAGG ccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTT CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA

 MRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGDYKDDDDK

purify PCR fragment using Qiagen gel purification kit

Use 1 ug for IVT Purify PCR fragment using Qiagen gel purification kit

Use 1 ug for IVT

[[File:2683 PCR for mRNA.JPG|RTENOTITLE]]

purify PCR fragment

use 1 ug for IVT

== Add an EGFP tag to Flag tag Clone ==

'''EGFP PCR template '''

T7-EGFP-F TAATACGACTCACTATAAGGAGACCATGGTGAGCAAGGGC

EGFP-R tttttttTTTTTTTTTTTTTTTTACTTGTACAGCTCGTCC (GGACGAGCTGTACAAGTAAAAAAAAAAAAAAAAaaaaaaa)

'''SARS2-EGFP fusion PCR template'''

clones should already have a Flag tag

to PCR out clone, such as 2686, 2689, 2690, 2691 original IDT fragments in pJet1.2

'''T7-mRNA-F4'''

TAATACGACTCACTATAAGGAGgccACCATG

Flag-EGFP-R ctcctcgcccttgctcaccttgtcgtcatcgtctttgtag and PCR EGFP from 2743 pLVX-Spike-IRES-EGFP

Flag-EGFP-F ctacaaagacgatgacgacaaggtgagcaagggcgaggag

EGFP-R tttttttTTTTTTTTTTTTTTTTACTTGTACAGCTCGTCC (GGACGAGCTGTACAAGTAAAAAAAAAAAAAAAAaaaaaaa)

gel purified two fragments and combine

PCR again using 

'''T7-mRNA-F4'''

EGFP-R tttttttTTTTTTTTTTTTTTTTACTTGTACAGCTCGTCC (GGACGAGCTGTACAAGTAAAAAAAAAAAAAAAAaaaaaaa)

purify and use it as template for IVT

transfection 293T cells with purified mRNA  to see GFP expression

== For amplifying DNA template from pCDNA5-FRT ==

T7-mRNA-F2

ATTAATACGACTCACTATAAGGAGACCCAAG

pCDNA5-mRNA-R TTTTTTTTTTTTTTTTTTTTGGGGTCGAGGTGCCGTAAAG (CTTTACGGCACCTCGACCCCAAAAAAAAAAAAAAAAAAAA)

== Current Working Protocol  ==

=== mRNA for transfection in vitro ===

Template preparation by PCR using 2X Taq PCR mix

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Clone # 
| F Primer 
| R Prime
| PCR Length
|-
| 
| 
| 
| 
|-
| 
| 
| 
| 
|-
| 
| 
| 
| 
|-
| 
| 
| 
| 
|}

After PCR, take 5 ul to check purity on agarose gel.  Purify the remaining PCR product using a spin column, elute DNA using 25 ul ddH2O, measure DNA concentration using nanodrop

=== mRNA as vaccine for animal study ===

Template preparation by PCR using 2X Taq PCR mix

{| border="1" cellspacing="1" cellpadding="1" style="width: 577px;"
|-
| Clone # 
| Name
| Des
| F Primer 
| style="width: 165px;" | R Prime
| style="width: 37px;" | PCR Length
|-
| 2684 
| DS2
| T7-IRES-DS2
|
T7-IRES-F

or T7-F

or IVT-T7-F

| style="width: 165px;" | Flag-R 
| style="width: 37px;" | 997
|-
| 2838
| DS2
| T7-DS2 in pJet
| T7-DS2-F 
| style="width: 165px;" | Flag-R
| style="width: 37px;" | 454
|-
| 2831 
| DS2-fd
| T7-DS2-fd in pJet
| T7-DS2-F 
| style="width: 165px;" | Foldon-R 
| style="width: 37px;" | 500
|-
| 2823
| DS2
| T7-DS2 in pJet 
| T7-DS2-F 
| style="width: 165px;" | Flag-R 
| style="width: 37px;" | 454 
|-
| 2823
| DS2 SAM
| T7-DS2 in pT7-VEE 
| T7 or T7-VEE-F
| style="width: 165px;" | Flag-R or VEE-pA-R 
| style="width: 37px;" | 8025
|-
| 2831
| DS2-fd SAM
| T7-DS2-fd in pT7-VEE
| T7 or T7-VEE-F
| style="width: 165px;" | Foldon-R or VEE-pA-R 
| style="width: 37px;" | 8065
|-
| 2874 
| RBD-fd
| Full length RBD fuse to fd, with polyA
| 
| style="width: 165px;" | 
| style="width: 37px;" | 
|}

Flag-R TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG

(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)

T7-DS2-F TAATACGACTCACTATAAGGAGGCCACCATGGTCAGACAAATC

Foldon-R tttttttttttttttttttttacaagaaggtcgacagcag

(ctgctgtcgaccttcttgtaaaaaaaaaaaaaaaaaaaaa)

VEE-pA-R  GAATTAATTCCCCTCGAcgcgttctag

T7-VEE-F TAATACGACTCACTATA'''''A'''G''ATGGGCGGCGCATGAGAG

After PCR, take 5 ul to check purity on agarose gel.  Purify the remaining PCR product using a spin column, elute DNA using 25 ul ddH2O, measure DNA concentration using nanodrop

[[File:DS2 DS2fd PCR.JPG|RTENOTITLE]]

[[File:2838 2831 PCR1.JPG|RTENOTITLE]]

[[File:2838 2831 PCR2.JPG|RTENOTITLE]]

== Use EMCV IRES to replace cap ==

PCR EMCV IRES from 959,961,960

T7 promoter: TAATACGACTCACTATAGGG

EMCV-IRES-R GGTGGTGGCCATATTATCATC

PCR from GOI 2684 pJet1.2-SARS2-S1-2

Spike RDB DS2

F: GATGATAATATGGCCACCACCCATATGGTCAGACAAATCGCTCCAG

R: 

== Make Self-amplifying mRNA (SAM) using VEE vector ==

PCR amplify

== PCR making template from pCAGGS vector ==

for making mRNA PCR using pCAGGS-T7 TAATACGACTCACTATAAGcattttggcaaggccaccatg pCAGGS-pA tTTTTTTTTTTTTTTTTTTTTTCATGGCAGCCAGCATATG (CATATGCTGGCTGCCATGAAAAAAAAAAAAAAAAAAAAAa)

template: 2725 pCAGGS-SARS2-RBD or 2754 pCAGGS-SARS2-S-6H

== mRNA from 2874 pCDNA5-frt-SARS2-RBD-fd  ==

RBD trimer, signal peptide, Codon optimized, 190 poly A tail

=== 1, PCR ===

primers

T7-mRNA-F2 ATTAATACGACTCACTATAAGG AGACCCAAG

2874-R AACGGGCCCTCTAGActcgAGAATTAATTCCCCTCGAcgc (gcgTCGAGGGGAATTAATTCTcgagTCTAGAGGGCCCGTT)

extension time 1 min

precipitate using isopropanol

optionally, remove extra nt from 3' using PacI or Xho1 

=== 2, Digestion plasmid DNA with <s>PacI, or Xho1 or</s> MluI or XbaI ===

PacI digested template has many large RNA or DNA bands,

For T7-VEE, PacI  and XhoI produces 3´overhang, so they are not good for linearization, if PacI or Xho1 is to be used, DNA template must be blunted by  adding DNA Polymerase I Large (Klenow) Fragment at a concentration of 5u/μg and incubate the reaction mixture for 15 minutes at 22°C.  

'''For  digestion of 36 ug of T7-VEE plasmid in 200 μL'''

36 μL T7-VEE Plasmid (1 μg/μL)

20 μL CutSmart Buffer (10X)

139 μL Sterile distilled water

5 μL MluI-HF (10,000 units/mL, NEB)

Incubate digestion mixtures at 37°C for 3 hours.

Add the same volume (200 μL) of phenol/chloroform/isoamyl alcohol (25:24:1) to each eppendorf tube containing the digestion mixture. Vortex the eppendorf tubes for 60 seconds. Centrifuge at high speed (14,000 rpm) for 5 minutes. Transfer each aqueous phase (top layer) into new eppendorf tubes.

Note: T7-VEE plasmid is a large sized DNA and may be trapped in the interphase layer with the proteins. When the interphase is sticky (DNA is trapped), collect the interphase layer along with the aqueous layer. To remove the excess phenol, add an equal volume (200 μL) of chloroform/isoamyl alcohol (24:1) to each eppendorf tubes. Vortex the eppendorf tubes for 60 seconds and centrifuge at high speed (14,000 rpm) for 5 minutes. Transfer each aqueous phase (top layer) into new eppendorf tubes. Add 1/10 volume (20 μL) of 3 M sodium acetate, pH 5.2 (this can be found in the RiboMAX Large Scale RNA Production kit) and 2.5 X volume (500 μL) of 100% ethanol to each eppendorf tubes. Vortex and place at -80°C for 5 minutes or store at -20°C until use. Note: If there are no plans to proceed to RNA synthesis on the same day, stop at this step. DNA will be stable for several months at -20°C. Centrifuge at high speed (14,000 rpm) for 10-15 minutes at 4°C to pellet the DNA plasmid. Remove the ethanol carefully with a pipet tip, being careful not to dislodge the DNA pellet. Gently add 1 mL 70% ethanol to wash the DNA pellet. Centrifuge at high speed at 4°C for 1 minute. Remove the ethanol carefully with a pipet tip, being careful not to dislodge the RNA pellet. Centrifuge shortly, and carefully remove any visible residual ethanol with a P20 pipette. Drying the DNA pellet is not necessary. In general, approximately 20% of plasmid may be lost during purification. Resuspend the DNA pellet in a small volume (15~20 μL, depending on starting amount) of DNase & RNase-free water before measuring the concentration. Measure the DNA concentration of each linearized DNA plasmid. Adjust the concentration to 1 μg/μL.

'''mRNA analysis by agarose'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Desc
| Note
|-
| 1
| PacI digested plasmid, large scale
| need to remove DNA using DNase I
|-
| 2
| PacI digested plasmid, small scale 
| 
|-
| 3
| Xho1 digested plasmid, small scale
| 
|-
| 4
| PCR template, small scale
| 
|-
| 5
| DNA ladder, NEB, 1 Kb
| 
|-
| 6
| PCR template 1, precipitate with isopropanol + glycogen 
| 
|-
| 7
| PCR template 2, precipitate with isopropanol 
| 
|}

[[File:ShenDong mRNA QC 12 14 2020 long.JPG|RTENOTITLE]]

mRNA made from 6 + 7 combined, 4 tubes of 40 ul reaction from template 6, 4 tubes of 40 ul reaction from template 7, purified using Qiagen RNeasy miniprep kit

[[File:MRNA2.jpg|RTENOTITLE]]

CleanCap is used for easy one -step reaction, while enzymatic capping method requires three-step reaction but it minimizes the IFN responses by combination with phosphatase treatment

== High yield Production of mRNA ==

10 mL transcription reaction mixture 30 mM Tris (pH 8.1 at 37°C), 15 mM Mg2+ , 10 mM dithiothreitol (DTT), 2 mM spermidine, 0.01% (v/v) Triton X-100, 4 mM each NTP, 1 mL of PCR-generated DNA template, and 0.1 mg/mL of T7 RNAP

optimal concentration of Mg2+ is 15 ~45 mM After 2.5 h of incubation at 37°C, pyrophosphate, which forms during in vitro transcription reaction, was pelleted down by centrifugation, and additional Mg2+ should be added to the reaction.  The reaction continued till 5 h.  Size-exclusion FPLC based RNA purification RNA sample preparation for chromatographic purification Transcription mixtures for the S-box RNA were heated to 90°C for 5 min, immediately transferred to ice for 5 min and then pelleted down (centrifuged at 16, 000g for 10 min). The supernatant was collected and mixed as 1: 4 with Tris-EDTA buffer containing 8 M urea and 100 mM NaCl before loading onto size-exclusion FPLC (SEC) columns. Sample pre-load treatment was operated to remove the T7 RNAP, residual pyrophosphates and aggregates formed during in vitro transcription.

Denaturing chromatographic purification The glass columns (890/10 mm) and (xk16/1000 mm) were packed according to manufacturer’s specifications with gel filtration matrix (Superdex-75, Superdex-200) and equilibrated in a cold room (4°C) with several column volumes (CV) of specified buffer. The transcription mixture from the previous step was loaded directly onto the equilibrated SEC column, and the chromatography was performed at 1 mL/min flow rate, collecting 2 mL fractions. Fractions were analyzed by denaturing urea-PAGE. S-box RNA containing fractions were combined and concentrated to 0.5 mL using Millipore centrifugal filter units (EMD Millipore), washed twice with the appropriate buffer for chemical probing analysis. All experiments were performed at 4°C. The efficiency of proposed purification system was confirmed through SHAPE experiments for S-box RNA.

== Transfection ==

Thaw mRNAs were thawed on ice and denatured at 65°C for 3min

Incubation at room temperature for 45min.

'''1. Lipofectamine 3000'''

*On day 0, HEK 293T cells are seeded at a concentration of 5 × 105 cells/well in a 6-well plate to ascertain 70–90 % of confluency on the day of transfection (day 1).
*For transfection, a ratio of 2 μl of Lipofectamine® reagent to 1 μg of RNA is used. Cells are transfected with 1 and 2 μg of mRNA. Transfection is performed in duplicates.
*On the day of transfection, an appropriate volume of Lipofectamine® reagent is prepared in Opti-MEM® medium and incubated at room temperature for 5 min.
* 1 and 2 μg of mRNA are diluted in Opti-MEM® medium. If required, pre-dilute mRNA to a concentration of 0.5 μg/μl.
*Mix Lipofectamine® reagent and Opti-MEM®-diluted mRNA gently in a total volume of 500 μl at a 1:1 ratio (v/v) and incubated at room temperature for 20 min.
*Wash cells twice with 500 μl DMEM medium containing glutamine but without FBS. Add 2 ml of DMEM to the cells.
*Add 500 μl of mRNA–lipid complexes to each well. Mix gently. 
*Incubate cells at 37 °C in a CO2-incubator for 4–6 h.
*After the incubation, replace the medium with 2 ml of fresh DMEM medium containing 10 % FBS.
*

'''2. TT3 nanoparticles'''

lipid:DOPE:cholesterol:DMG-PEG2000 at a molar ratio of 20:30:40:0.75 ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869688/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869688/])

3. Protamine as an immunostimulatory component

Protamine. Pharmaceutical Protamine is available at two concentrations: 1000 (1 ml neutralizes 1000 IU of heparin) or 5000 (1 ml neutralizes 5000 IU of heparin). To generate particles of 130 nm, we use Protamine Ipex 5000 from MEDA Pharmaceuticals (Protamine hydrochloride 5000 IU/ml). The Protamine is stored at 4 °C 

Dissolve the protamine in Ringer lactate solution.  While stirring, slowly add the protamine-Ringer lactate solution half of the mRNA until you reach a weight ratio of mRNA and protamine of 2:1.  Stir the solution for another 10 min to ensure the formation of stable complexes.  Add the remaining half of free mRNA and stir briefly.  Adjust the final concentration of the vaccine with Ringer lactate solution.

Carry out all procedures at room temperature unless otherwise specified.

 This step is performed under laminar flow using sterile equipment.

*Prepare a 0.5 mg/ml Protamine solution (see Note 5) by diluting 10 μl of Protamine 5000 in 280 μl of water. 
*Prepare a 0.5 mg/ml RNA solution (see Note 5) by diluting the RNA stock with the appropriate amount of water.
*Add an equal amount of Protamine solution to the RNA solution (see Note 6), and pipet quickly up and down at least ten times (see Note 7).
*Leave the solution at room temperature for 10 min (see Note 8). 1. Dilute 40 μl of the Protamine–RNA formulation (containing 10 μg of RNA and 10 μg of Protamine) with 1 ml of water (see Note 9).

Set the viscosity of the analyzer to 0.89 (select within the software setting either measurement in water or a viscosity of 0.89). As shown in Fig. 1, particles made in the conditions described above are approximately 130 nm in size with a polydispersity index (PDI) of less than 0.3 (see Note 10).

Protamine sulfate, such as Valeant Protamine, gave identical results. Freezing Protamine solutions may jeopardize its functionality as far as production of immunostimulating RNA polyplex is concerned. Pharmaceutical Protamine should be stored at 4 °C as indicated on the package. Any RNA (oligonucleotide, long uncapped and/or non-polyadenylatedmRNA, or capped mRNA) can be used to generate the 130 nm particles

== Nonobody mRNA ==

HiScribe T7 ARCA mRNA kit (with tailing; NEB; #E2060S). PCR product should be purified and run on an agarose gel to confirm amplicon size prior to its use as a template in the T7 ARCA mRNA transcription reaction. Generally, 0.1–0.5 mg of PCR fragments can be used in a 20-mL in vitro transcription reaction. After the preparation of PCR templates, standard mRNA synthesis with modified nucleotides should be started with thawing the necessary kit components, mixing, and pulse spinning in a microfuge to collect solutions to the bottoms of the tubes. The reaction was assembled at room temperature in the following order: nuclease-free water (to 20 uL), 2 x ARCA/NTP mix (10 uL), 5mCTP (10 mM, 2.5 uL), pseudo-UTP (10 mM, 2.5 uL), template DNA (1 mg), and T7 RNA polymerase mix (2 uL); mixed thoroughly; and pulse spun in a microfuge. Incubation was at 37oC for 30 min. DNase treatment to remove template DNA included adding 2 uL of DNase I, mixing well, and incubating at 37 oC for 15 min. poly(A) tailing starts with setting up the tailing reaction as below. Standard tailing reaction volume was 50 uL: H2O (20 uL), IVT reaction (20 uL), 10 X poly(A) polymerase reaction buffer (5 uL), and poly(A) polymerase. It was mixed thoroughly, pulse spun in a microfuge, and incubated at 37oC for 30 min. Synthesized mRNA can be purified by LiCl precipitation, phenol/chloroform extraction, followed by ethanol precipitation. To the 50-mL tailing reaction, 25 mL LiCl solution was added and mixed well. Incubation at -20 oC for 30 min was completed, as was centrifugation at 4oC for 15 min at top speed to pellet the RNA. The supernatant was removed carefully, the pellet was rinsed by adding 500 mL of cold 70% ethanol and centrifuged at 4oC for 10 min, ethanol was removed carefully, the tube was spun briefly to bring down any liquid on the wall, residual liquid was removed carefully using a sharp tip, the pellet was air dried, the mRNA was resuspend in 50 mL of 0.1 mM EDTA or a suitable RNA storage solution, and the RNA was heated at 65oC for 5– 10 min to completely dissolve the RNA and mixed well.

== LNP ==

LNP were prepared by mixing ethanol and aqueous phase at a 1:3 volumetric ratio in a microfluidic device, using syringe pumps as previously described (Chen, Love et al., 2012). The ethanol phase was prepared by solubilizing a mixture of ionizable lipidoid cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), cholesterol (Avanti), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000] (ammonium salt) (C14-PEG 2000, Avanti) at a molar ratio of 35:16:46.5:2.5 The aqueous phase was prepared in 10 mM citrate buffer (pH 3) with either trastuzumab m 164 RNA or luciferase mRNA (Firefly luciferase mRNA, TranslateBio). LNPs were dialyzed against PBS in a Slide-A-Lyzer™ G2 Dialysis Cassettes, 20,000 MWCO (Thermo Fisher) for 2h at RT. The concentration of mRNA encapsulated into LNPs nanoparticles was analyzed using Quant-iT RiboGreen assay (Thermo Fisher), according to manufacturer’s protocol. The efficacy of mRNA encapsulation into LNPs was calculated by comparing measurements in the absence and presence of 1% (v/v) Triton X- 100. Nanoparticle size, polydispersity (PDI), and ζ-potential were analyzed by dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). LNP hydrodynamic diameters are reported in the volume weighting mode and are an average of three independent measurements (Supplemental Table 5).

== RNA capping using Vaccinia Capping Enzyme and Cap 2´-O-Methyltransferase  ==
<div>[https://pubmed.ncbi.nlm.nih.gov/27368341/ https://pubmed.ncbi.nlm.nih.gov/27368341/] </div><div>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753152/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753152/] </div><div> </div><div>Add 7 ul 32 mM SAM to 100 ul 10X Capping Buffer (Final SAM is 2 mM)</div><div> </div><div>Combine uncapped RNA and nuclease-free water in a final volume of 14.0 μl. (Refer to step 1 in the notes on use).</div><div> </div><div>Heat at 65°C for 5 minutes (Refer to step 2 in the notes on use).</div><div> </div><div>Place tube on ice for 5 minutes.</div><div> </div><div>Add the following components in the order specified:</div><div>Denatured uncapped RNA (from above) 14.0 μl</div><div>10X Capping Buffer with 2 mM SAM: 2.0 μl</div><div>GTP (10 mM) 1.0 μl</div><div><s>SAM (4 mM, dilute 32 mM stock to 4 mM) 1.0 μl</s></div><div>Vaccinia Capping Enzyme (10 U/μl) 1.0 μl</div><div>mRNA Cap 2´-O-Methyltransferase (50 U/μl)  1.0 μl</div><div>RNase Inhibitor, Murine 0.5 μl</div><div>Total: 20.0 μl</div><div>Note: Use of RNase Inhibitor is recommended to enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor (e.g., Murine RNase Inhibitor NEB #M0314) during reaction set up. Subtract the additional volume from the amount of H2O used in the reaction.</div><div> </div><div>Incubate at 37°C for 60 min </div><div>(For RNA less than 200 nt long increase incubation time to 2 hours).</div><div> </div><div> </div><div>RNase Inhibitor, Murine M0314S40 units/μl 3,000 units $73.00</div><div> </div>
IVT reactions: 1 µM primers, 40 mM Tris pH 8, 5 mM DTT, 1 mM spermidine, 0.01% Triton X-100, 4 mM each of NTP in the RNA sequence, 0.2 µM T7 RNA polymerase, 20–60 mM MgCl2.

=== RNA purification using anion-exchange chromatography ===

RNA from IVT can be purified using anion exchange chromatography on a 22 × 250 mm DNAPac PA100 column (Dionex) operated at 80°C. [https://www.thermofisher.com/order/catalog/product/043011#/043011 https://www.thermofisher.com/order/catalog/product/043011#/043011]

The column is run in buffers containing 20 mM Tris pH 8.0 and 5 M Urea (pH adjusted to 8.0 at room temperature). RNA of different lengths is eluted with a NaCl gradient. Salt gradients can be designed according to the respective RNA lengths to be separated. Short RNAs (2–10 nt) typically elute between 50 and 200 mM NaCl while longer constructs (60–80 nt) elute at 300–400 mM NaCl. RNA containing fractions can be analyzed using urea-PAGE, pooled and precipitated  as below. After precipitation, RNAs can be desalted using a prepacked PD-10 Desalting column (GE Healthcare) and subsequently concentrated using a speedvac device.

=== RNA precipitation ===

RNA can be precipitated with 0.3 M NaOAc pH 5.2 and 0.7 volumes of isopropanol at −20°C for at least 2 h. The reaction is then centrifuged at 9000g and −5°C for 1 h and the pellet is washed with cold 75% ethanol and centrifuged again for 30 min. The resulting pellet is air dried and resuspended in the desired buffer for further  applications. For short RNAs (2–10 nt), precipitation can be carried out with 0.2 M NaCl and 3.5 volumes of EtOH (replacing the NaOAc and isopropanol) and a precipitation time of 12 h

=== RNA capping ===

Capping reactions is performed in buffer containing 50 mM Tris, pH 8, 5 mM KCl, 1 mM MgCl2, and 1 mM DTT with 20 µM RNA. 0.5 mM GTP, 0.1 mM SAM, and capping enzyme according to the previously determined substrate to enzyme ratio is added. The reaction is incubated at 37°C and prepared for analysis by Urea-PAGE by adding an equal volume of sample buffer containing 8 M Urea, 20 mM EDTA, 2 mM Tris pH 8, and Bromophenol blue and Xylene Cyanol.

For cap-1:  cap-1 RNA, Vaccinia MTase (NEB) is added to the capping reaction

RNA promoter sequence Promoter sequence T7 promoter Φ6.5 (for start with G) CGTAATACGACTCACTATAGG T7 promoter Φ2.5 (for start with A) CGTAATACGACTCACTATTAG

== Cas13a ==

In vitro transcription of Cas13a. Plasmids were linearized with Not-I HF (New England Biolabs) overnight at 37 °C. Linearized templates were purified by sodium acetate (Thermo Fisher Scientific) precipitation and rehydrated with nuclease-free water. In vitro transcription was performed overnight at 37 °C using the HiScribe T7 Kit (NEB) following the manufacturer’s instructions (N1-methyl-pseudouridine modified). The resulting RNA was treated with DNase I (Aldevron) for 30 min to remove the template, and it was purified using lithium chloride precipitation (Thermo Fisher Scientific). The RNA was heat denatured at 65 °C for 10 min before capping with a Cap-1 structure using guanylyl transferase and 2′-O-methyltransferase (Aldevron). mRNA was then purified by lithium chloride precipitation, treated with alkaline phosphatase (NEB) and purified again. mRNA concentration was measured using a Nanodrop. mRNA stock concentrations were 1–3 mg ml−1. Purified mRNA products were analyzed by gel electrophoresis to ensure purity. crRNA guides were purchased from Integrated DNA Technologies or GenScript [https://www.aldevron.com/hubfs/content/Documents/ald-IVT-enzyme-tech-guide-090010916.pdf https://www.aldevron.com/hubfs/content/Documents/ald-IVT-enzyme-tech-guide-090010916.pdf]

== Enzymes for mRNA research ==

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986899/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986899/]

T7 RNA Polymerase 9135-130MCG 1

2598 pET29-Turbo-T7-RNAPo

E coli Inorganic Pyrophosphatase 9132-350MCG 1

2259 need to do cloning

DNase I 9136-70MCG 1 

202 pET28-DNase1 need to subclone into pMBP vector

Ribonuclease Inhibitor 9134-180MCG 1

[http://18.206.161.119/eln/index.php/623_625_637_1494_1499_1734_1735_1740_1747_1750 pT7A30-RNaseInhibitor Sus scrofa (wild pig) RNase Inhibitor 1499] 

Guanylyltransferase 9131-300MCG 2

2'-O-Methyltransferase 9130-1450MCG 2 An E. coli strain that carries the gene for the Vaccinia mRNA Cap 2´-O-Methyltransferase. Poly(A) Polymerase 9133-430MCG 2

Ribonuclease Inhibitor 9134-660MCG 2

Guanylyltransferase Catalyzes addition of 7-methylguanylate cap structure (Cap 0) to the 5’ end of RNA in three steps:

<div>T7 RNA Polymerase 2598 pET29-Turbo-T7-RNAPo</div><div>E coli Inorganic Pyrophosphatase  2259  </div><div>Ribonuclease Inhibitor  pT7A30-RNaseInhibitor Sus scrofa (wild pig)  RNase Inhibitor 1499 </div><div>Guanylyltransferase  2943</div><div>2'-O-Methyltransferase 2753</div><div>E coli  Poly(A) Polymerase    2773</div><div>Yeast Poly(A) Polymerase  940 pET28b-yPolyA-Pol</div><div> </div><div> </div>
2’-O-Methyltransferase Reactions 1 & 2: Converts Cap 0 to Cap 1 by adding a methyl group to the ribose 2’-OH of the first nucleotide adjacent to the cap structure. Activity can be measured using the EPIgeneousTM Methyltransferase Assay (Cisbio).

Reaction 3: Conversion of an RNA substrate pppN(pN)n to GpppN(pN)n with increasing amounts of enzyme can be visualized by gel electrophoresis (uncapped, lower band; capped, upper band).

Methyl transfer from SAM to GpppN(pN)n producing SAH and Cap 0 mRNA can be measured using the EPIgeneousTM Methyltransferase Assay (Cisbio).

[https://www.aldevron.com/products/ivt-enzyme-kits https://www.aldevron.com/products/ivt-enzyme-kits]

IVT Buffer: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306405/pdf/nar00265-0210.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306405/pdf/nar00265-0210.pdf]

40 mM Tris-HCl (pH 8.1 at 37°C), 1 mM spermidine, 5 mM dithiothreitol, 50 ug/ml bovine serum albumin, 0.01% (vlv) Triton X-100 and 80 mg/ml polyethylene glycol (8000 MW)

[https://www.neb.com/gmp/gmp-grade-reagents-for-rna-synthesis https://www.neb.com/gmp/gmp-grade-reagents-for-rna-synthesis]

Inorganic pyrophosphatase [https://www.neb.com/products/m0361-pyrophosphatase-inorganic-e-coli https://www.neb.com/products/m0361-pyrophosphatase-inorganic-e-coli] PPase is prepared from an E. coli strain containing a clone of the E. coli inorganic pyrophosphatase gene.

DNaseI : [https://www.neb.com/products/m0303-dnase-i-rnase-free https://www.neb.com/products/m0303-dnase-i-rnase-free] An E. Coli strain that carries an MBP fusion clone of Bovine Pancreatic DNase I.

== Flp mRNA ==

PCR  using these two primers

Template; 2895 T7-VEE-NLS-Flp

T7-HBG-F TAATACGACTCACTATAAGGACATTTGCTTCTGACACAAC VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

sequence

T7 (modified) TAATACGACTCACTATAAGG   ACATTTGCTTCTGACACAAC TGTGTTCACTAGCAACCTCAAACAATT Kozak GCCACC ATGGCCCCCAAGAAAAAGCGGAAAGTGATGAGCCAGTTCGACATCCTGTGCAAGACCCCCCCCAAGGTGCTCGTGCGGCAGTTCGTGGAAAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCTCTTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGCGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAAGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTTACCATCATCCCCTACAACGGCCAGAAGCACCAGAGCGACATCACCGATATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAAGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGAGATCACAGAGAAGATCCTGAACAGCTTCGAGTACACCAGCCGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACCTTTATCAACTGCGGCCGGTTCTCCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTCGTGACCGAGACAAAGACCAGCGTGTCCCGGCACATCTACTTTTTCAGCGCCAGAGGCCGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGAAACAGCGAGCCCGTGCTGAAGAGAGTGAACCGGACCGGCAACAGCAGCTCCAACAAGCAGGAATACCAGCTGCTGAAGGACAACCTCGTGCGGTCCTACAACAAGGCCCTGAAGAAAAACGCCCCCTACCCCATCTTCGCCATTAAGAACGGCCCCAAGTCCCACATCGGCCGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACAGAGCTGACCAACGTCGTGGGCAATTGGAGCGACAAGAGGGCCTCTGCCGTGGCCAGAACCACCTACACCCACCAGATCACAGCCATCCCCGACCACTACTTCGCCCTGGTGTCTCGGTACTACGCCTACGACCCCATCAGCAAAGAGATGATCGCCCTGAAGGACGAGACAAACCCCATCGAGGAATGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCTGCCTGGAACGGCATCATCTCCCAGGAAGTGCTGGACTACCTGAGCAGCTACATCAACCGGCGGATCTAG

Not1  GCGGCCGC AAGCTGCTTACATAGAACTCGCGGCGATTG Sph1 GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA PacI  ttaattaa

Xba1 tctaga

Mlu1 acgcgT CGAGGGGAATTAATTC  (VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag )
[[Category:Pages with broken file links]]

== EGFP mRNA Protocol ==

to make template for Cleancap by PCR

T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

T7-HBA2-F is not as good as T7AG-HBG-F  

Template: '''2885 '''T7-VEE-EGFP

(F primer T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC- for cleancap)

sequence, 1113 bp

GTTAATACGACTCACTATAag TCTTCTGGTCCCCACAGACTCAGAGAGAACCC Kozak gccacc

EGFP atggtgTCTaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa Not1 GCGGCCGC AAGCTGCTTACATAGAACTCGCGGCGATTG

Sph1

GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

PacI

ttaattaa

Xba1

tctaga

Mlu1

acgcgT CGAGGGGAATTAATTC

(R primer VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag )

=== Cy5-UTP ===

[[File:Cy5-UTP.png|RTENOTITLE]]

IVT: Use 1 ul cy5-UTP for 80 ul reaction, 4 ul of buffer , 4 ul of each NTP, 4 ul enzyme, react 24 hours

[https://www.apexbt.com/cy5-utp.html, 25ul https://www.apexbt.com/cy5-utp.html, 25ul] (10mM) $165.00, Cy5-UTP Catalog No. B8333

[https://www.perkinelmer.com/product/cyanine-5-utp-nel583001ea Cyanine https://www.perkinelmer.com/product/cyanine-5-utp-nel583001ea Cyanine] 5-UTP, 100 nmol, NEL583001EA 577.00 USD

[https://www.enzolifesciences.com/ENZ-42506/cyanine-5-utp-enhanced/ ENZ-42506 https://www.enzolifesciences.com/ENZ-42506/cyanine-5-utp-enhanced/  ENZ-42506] 250 nmol 344.00 USD

[https://promocell.com/product/cy5-utp/ PK-PFCY5-UTP-10 https://promocell.com/product/cy5-utp/ PK-PFCY5-UTP-10] 10 µl $ 299.00

[https://www.trilinkbiotech.com/cyanine-5-aminoallyluridine-5-triphosphate.html https://www.trilinkbiotech.com/cyanine-5-aminoallyluridine-5-triphosphate.html]

[https://www.cytivalifesciences.com/en/us/shop/molecular-biology/nucleic-acid-microarrays-and-accessories/amersham-cydye-fluorescent-nucleotides-p-06187#order https://www.cytivalifesciences.com/en/us/shop/molecular-biology/nucleic-acid-microarrays-and-accessories/amersham-cydye-fluorescent-nucleotides-p-06187#order]

PA55026Cy5-UTP 100 nmol 598.00 USD

[https://www.scbt.com/p/cyanine-5-utp Cyanine https://www.scbt.com/p/cyanine-5-utp Cyanine] 5-UTP, 250 nmolsc-499469,  250 nmol $316.00

[[File:2885 cy5-EGFP-mRNA.jpg|RTENOTITLE]]

[[File:2885 cy5-EGFP-mRNA 2.jpg|RTENOTITLE]]

=== NEB Protocol: ===

4 ul of each: 100 mM ATP, GTP, UTP, CTP, 3.2 ul of 100mM Cleancap, 4 ul of 10X Buffer, 40 ul of 2885 PCR product (2 ug total), 1 ul of 10mM Cy5-UTP (santa cruz), 5 ul of T7 RNA Polymerase Mix, Water to 80 ul, reaction for 24 hours.

Purify using RNAeazy spin column

Yield:  150 ul of 1.7 ug/ul, 50 ul of 816ng/ul

Total yield:  255 ug + 40 ug= 295 ug

=== Lucigen AmpliScribe High Yield Transcription Kits Protocol ===

x µL Nuclease-Free Water,

Sterile 1 µg linearised template DNA with T7 promoter

2 µL AmpliScribe T7 10X Reaction Buffer

1.5 µL 100 mM ATP

1.5 µL 100 mM CTP

1.5 µL 100 mM GTP

1 µL 100 mM UTP

6 µL 4 mM Cy5-UTP

2 µL 100 mM DTT

0.5 µL RiboGuard RNase Inhibitor

2 µL AmpliScribe T7 RNA Polymerase

20 µL Total reaction volume

Incubate at 37 °C for 2 hours

== ==

MRNA by In vitro Transcription

2022-02-13T15:44:44Z

Paul.xu:

== Linearization of plasmid DNA ==

Linearize 10 ug of plasmid DNA in a 100 uL restriction enzyme digestion as follows:

#Reaction Assembly, a total of 100 uL:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | Reagents
| bgcolor="#eaeaea" | Volume (uL)
|-
| bgcolor="#ffffff" | H20
| bgcolor="#ffffff" | 70
|-
| bgcolor="#eaeaea" | 10 x Buffer
| bgcolor="#eaeaea" | 10
|-
| bgcolor="#ffffff" | DNA (10 ug)
| bgcolor="#ffffff" | 10 (For 1ug/uL DNA)
|-
| bgcolor="#eaeaea" | Enzyme
| bgcolor="#eaeaea" | 10
|-
! bgcolor="#dadada" | Total
! bgcolor="#dadada" | 100
|}

#Digest 3 hours at 37° (or whatever temperature the restriction enzyme requires).
#Run out 0.5 ug (5 uL) of linearized DNA with a size standard to confirm complete digestion.

== Purification of Linearized DNA ==

To recover the linearized plasmids with Qiagen MinElute PCR Purification Kit:

#Add 475 uL of Buffer PBI to 95 uL of remaining linearization reaction mix.
#Place in a MinElute column in a 2 mL collection tube.
#To bind DNA, apply the sample to the QIAquick column and centrifuge for 30 seconds at 13,200rpm.
#Wash MinElute column by adding 750 uL of Buffer PE and centrifuge for 30 seconds at 13,200rpm.
#Discard the flow-through and place the MinElute column back into the same tube, which should be empty. Centrifuge for an additional minute at 13,200rpm to remove any last traces of wash buffer.
#Place MinElute column in a clean 1.5 mL micro centrifuge tube
#To elute DNA, add 38 uL of Water to MinElute membrane, let column stand for 1 minute and centrifuge the column for 30 seconds at 13,200rpm.
#Quantify 1.5 uL DNA using Nanodrop©!.

== mRNA Making ==

To sythesize mRNA from linearized plasmids using Ambion messenger SP6 kit:

#Assemble the reaction at room temperature:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | Reagents
| bgcolor="#eaeaea" | Volume (uL)
|-
| bgcolor="#ffffff" | DNA (1 ug)
| bgcolor="#ffffff" | 4 (For 0.25 ug/uL DNA)
|-
| bgcolor="#eaeaea" | H20
| bgcolor="#eaeaea" | 2
|-
| bgcolor="#ffffff" | 2 x NTPs
| bgcolor="#ffffff" | 10
|-
| bgcolor="#eaeaea" | 10 x buffer
| bgcolor="#eaeaea" | 2
|-
| bgcolor="#ffffff" | Enzyme (SP6 and
RNasin)

| bgcolor="#ffffff" | 2
|-
! bgcolor="#dadada" | Total
! bgcolor="#dadada" | 20
|}

#Incubate at 37° for 2 hours in the incubator.
#Add 1 uL of DNase for 10 minutes at 37° to digest remaining DNA.

= mRNA Synthesis with Modified Nucleotides =

Modified mRNAs containing 5mCTP and Pseudo-UTP have been shown to suppress RNA-mediated innate immune activation in vivo. The HiScribe T7 ARCA mRNA Kit is capable of incorporation of 5mCTP and Pseudo-UTP, into mRNA. Up to 2.5 mM total modified nucleotides can be added into the transcription reaction without impacting the mRNA yield significantly. Other modified UTP or CTP may also be used but RNA yield will vary depending on the properties of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Please note modified nucleosides are not supplied with the kit. The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP. 

#Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order: 
{| border="0" width="100%" cellspacing="0" cellpadding="0"
|-
| Nuclease-free water
| to 20 µl
|-
| 2X ARCA/NTP Mix
| 10 µl
| 1 mM GTP, 4 mM ARCA, 1.25 mM CTP, 1.25 mM UTP, >1.25 mM ATP final
|-
| 5mCTP, 10 mM
| 2.5 µl
| 1.25 mM 5mCTP final
|-
| Pseudo-UTP, 10 mM
| 2.5 µl
| 1.25 mM Pseudo-UTP final
|-
| Template DNA
| X µl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 µl
|-
| Total reaction volume
| 20 µl
|}

#Follow standard mRNA synthesis protocol.

== mRNA Purification ==

To harvest the RNA using Qiagen MinElute RNeasy Protocol For RNA cleanup:

#Adjust the sample to a volume of 100 uL with RNase-free water, add 350 uL of RLT and mix thoroughly (note: all older kits require the addition of β-ME to buffer RLT)
#Add 250 uL of ethanol (96-100 %) to the diluted RNA, and mix thoroughly by pipetting. Do not centrifuge.
#Apply sample to an RNeasy mini column placed in a 2 mL collection tube. Close the tube gently, and centrifuge for 30 s at 13,200rpm. Discard flow-through and collection tube.
#Transfer the RNeasy column into a new 2 mL collection tube. Pipet 500 uL Buffer RPE onto the RNeasy column. Close tube gently, and centrifuge 1 minute for 13,200 rpm to wash the column and discard the flow-through.
#Add 500 uL of 80% Ethanol to RNeasy column. Close tube gently and centrifuge for 1 minute at 13,200 rpm. Discard flow-through and collection tube.
#Transfer the RNeasy column into a new 2 mL collection tube. Close tube gently and centrifuge for 5 minute at 13,200 rpm to dry the RNeasy silica-gel membrane.
#To elute transfer the RNeasy column to a new 1.5 ml collection tube. Pipet 50uL of RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube and centrifuge for 1 minute at 13,200 rpm.
#Immediately put collection tubes on ice, take 2 uL + 1.5ul of RNA out into two new 1.5 mL tubes.
#Quickly store the remaining RNA in -80℃.
#Run the taken-out 2 uL RNA on a gel and Quantify the 1.5 uL RNA using Nanodrop©!.
#Stored RNA can be diluted as necessary.
[[Category:Protocols]]

2951

2022-02-10T16:18:45Z

Paul.xu:

Cloing method: digest 2794 with Ega1, religate and transform screen plasmid using BglII

[[File:2951 EagI digestion.JPG|RTENOTITLE]]

2951 digestion 

[[File:2951 bglII.JPG|RTENOTITLE]]
[[Category:Cloning]]

Plasmid buffers

2022-01-23T16:32:55Z

Paul.xu:

Homemade Buffers for plasmid kits

== '''Mini-prep''' ==

 '''Buffer P1''' 50 mM Tris HCl, pH 8.0, 10 mM EDTA, (or 2. 5 ml of 100X Tris-EDTA buffer from Sigma #T-9285 in 50 ml water) 100 ug/ml RNase A.

'''P1''' from another protocol: 10 mM Tris-HCl, pH8, 100 ug/ml RNase A.

6.06 g Tris Base, 3.72 g Na2EDTA.2H2O in 800 ml H2O, adjust the pH with HCl, add water to 1 L, add 100 mg RNase A per liter of P1.

add some lyseblue (Thymophthalein)

'''Resuspension Buffer''': 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 ug/mL RNase A '''Lysis Buffer''': 200 mM NaOH, 1% SDS '''Precipitation Buffer''': 4.2 M guanidine hydrochloride, 0.9M Potassium acetate pH 4.8 '''Equilibration Buffer''': 7M Gu-HCl pH 5.5 '''Wash 1 Buffer''': 5M guanidine HCl, 30% Ethanol, 10 mM TRIS-HCl pH 6.6 '''Wash 2 Buffer''': 10 mM TRIS-HCl pH 7.5, 80% Ethanol '''Elution Solution''': Water '''RNase A''': Dissolve RNASE A powder in H2O to give 100 mg/ml. Boil 5 min to kill DNase. Aliquot and store at -20 oC. '''Buffer P2''' (200mM NaOH, 1% SDS): 1M NaOH 10 ml 10% SDS 5 ml Water 35 ml

Dissolve 8.0 g NaOH in 950 ml dH2O, 50 ml 20% SDS (w/v) solution.  The final volume should be 1 liter. Some protocols use 2% SDS.

'''Buffer P3'''

Dissolve 294.5 g potassium acetate in 500 ml dH2O.  Adjust the pH to 5.5 with glacial acetic acid (~110 ml).  Adjust the volume to 1 liter with dH2O.

3.0 M potassium acetate pH 5.5 can be stored either at room temp or 2~8°C

'''PB'''

5 M guanidine hydrochloride (MW 95.53 ), 20 mM Tris-HCl, pH 6.6, (25 oC), add ethanol to 38%.

200 ml:

95.53 g guanidine hydrochloride

8 ml of 0.5M Tris-HCl, pH6.6

76 ml of ethanol

'''PB from another protocol:''' 5 M Gu-HCl, 30% <strike>isopropanol</strike> ethanol

 '''Buffer DP3''' (for Qiagen Directprep 96-well miniprep)

3.0 M ammonium acetate pH 5.5

'''PB Buffer''': 5 M guanidine hydrochloride, 30%<strike>isopropanol</strike>.ethanol

'''Buffer FWB'''

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH2O.  Adjust the pH to 7.0 with NaOH.  Add 150 ml pure isopropanol and adjust the volume to 1 liter with dH2O.

'''Buffer QBT'''

Dissolve 43.83 g NaCl, <strike>10.46 g MOPS (free acid) .</strike>  7g Tris-HCl, 0.5 g Tris Base in 800 ml dH2O. <strike>Adjust the pH to 7.0 with NaOH. </strike> Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with dH2O.

750 mM NaCl 50 mM <strike>MOPS </strike>Tris-HCl, pH 7.0 15% isopropanol (v/v) 0.15% Triton® X-100 (v/v)

'''N3-Ca''': N3 with 1.4 M CaCl2 for precipitate high molecular weight RNA

'''2X PEG6000-NaCl2''' , 20% PEG 6000, 1.6 M NaCl.  for precipiate plasmid DNA: 4 ml of 50 % PEG 6000, 4 ml of 4 M NaCl, 2 ml H2O, autoclave.

'''Buffer QC'''

Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O.  Adjust the pH to 7.0 with NaOH.  Add 150 ml pure isopropanol.  Adjust the volume to 1 liter with dH2O.

1.0 M NaCl 50 mM MOPS pH 7.0 15% isopropanol (v/v)

'''Buffer QF'''

'''From Qiagen website'''

Dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml dH2O and adjust the pH to 8.5 with HCl.  Add 150 ml pure isopropanol.  Adjust the volume to 1 liter with dH2O.

1.25 M NaCl 50 mM Tris·Cl pH 8.5 15% isopropanol (v/v)

Store at 15–25°C

73.05 g NaCl

2.21g Trizma HCL

4.36 g Trizma Base

'''Buffer QN'''

'''from Qiagen website'''

Dissolve 93.50 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O and adjust the pH to 7.0 with NaOH.  Add 150 ml pure isopropanol.  Adjust the volume to 1 liter with dH2O.

1.6M NaCl 50 mM MOPS pH 7.0 15% isopropanol

'''Buffer STE'''

Dissolve 5.84 g NaCl, 1.21 g Tris base, and 0.37 g Na2EDTA*2H2O in 800 ml dH2O.  Adjust the pH to 8.0 with HCl.  Adjust the volume to 1 liter with dH2O.

 '''Buffer P3''' 3.0 M KOAc, pH 5.5 (29.5 g/ 100ml water, adjust pH with glacial acetic acid )

'''N3 from another protocol''':

4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to pH4.2~4.8 by adding acetic acid.

200 ml:

80 g guanidine hydrochloride

18g potassium acetate

10 ml acetic acid (add this inside the hood),

add DI water to dissolve

add water to 200 ml.

Yet another protocol

 4M guanidine hydrochloride (GuHCl) 0.5M potassium acetate pH 4.2

'''Improved N3''' 4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to pH4.2~4.8 by adding acetic acid.

200 ml:

80 g guanidine hydrochloride

18g potassium acetate

10 ml acetic acid (add this inside the hood),

'''6 ml of 1 M MgCl2'''

'''CaCl2 1.4 M'''

add DI water to dissolve

add water to 200 ml.

Buffer N3

4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8 Buffer PB

5 M Gu-HCl 30% isopropanol Buffer QG

5.5 M guanidine thiocyanate (GuSCN) 20 mM Tris HCl pH 6.6 Buffer PE

10 mM Tris-HCl pH 7.5 80% ethanol

'''Buffer QXB''' (for binding of large >3000 bp fragments to columns)

5 M GuHCl

 '''Buffer PE''' 80% EtOH. 25 mM NaCl, 5 mM Tris-HCl, pH 7.5

5 ml autoclaved Tris-HCl, pH 8.0

6 ml 4 M autoclaved NaCl

Autoclaved dd H2O to 250 ml

Add 800 ml EtOH '''Buffer EB''' 10 mM Tris·Cl, pH 8-8.5 make up to 1 liter with dH20

== '''Maxiprep''' ==

 '''QC buffer''' (wash buffer)

1.0M NaCl 50 mM MOPS pH 7.0 15% isopropanol

to make 2 L 666 ml 3M NaCl 200 ml 0.5 M MOPS pH 7.0 300 ml EtOH dH2O to 2 liters check the pH and adjust to 7.0 (it should be close) with NaOH or HCl

'''QF buffer''' (elution buffer)

1.25M NaCl 50 mM Tris-HCl pH 8.5 15% isopropanol

to make 2 L 833 ml 3M NaCl 100 ml 1M Tris pH 8.5 300 ml EtOH dH2O to 2 liters check the pH and adjust to 8.5 (it should be close) with NaOH or HCl store QC and QF in tightly capped containers at room temp.

'''Buffer QN'''

1.6M NaCl 50 mM MOPS pH 7.0 15% isopropanol

'''Buffer FWB2'''

1M potassium acetate, pH 5.0

'''QBT buffer'''

750 mM NaCl 50 mM MOPS pH 7.0 15% isopropanol 0.15% Triton X-100

to make 2 L: 500 ml 3M NaCl 200 ml 0.5 M MOPS pH 7.0 300 ml EtOH 3 ml Triton X-100 dH2O to 2 liters

Buffer QBT (equilibration buffer)

'''P1 buffer''' 1M Tris HCl pH 8.0 2.5 ml 0.5 M EDTA pH 8.0 1 ml 10 mg/ml RNase A, boiled 0.5 ml dH2O 46 ml 50ml total '''P2 buffer''' 2M NaOH 5 ml 20% SDS 2.5 ml dH2O 42.5 ml 50 ml total Apparently this solution can go bad due to oxidation: keep the container tightly capped. '''P3 buffer''' potassium acetate 25 g glacial acetic acid 13 ml H2O 87 ml 100 ml total final pH should be ~5.5

== Silica membrane, low endotoxin ==

'''Binding Solution''': 5% n-octyl-β-D-thioglucopyranoside, 8M guanidine hydrochloride.

Add 9 ml to 36 ml clear lysate (12 ml P1, 12 ml P2, 12 ml P3)--> Final conc is 1.6 M of guanidine hydrochloride, alternatively, use N3 containing 4.8 M guanidine hydrochloride. If N3 is used, wash column with Low-endotoxin Wash before washing with PB and PE.

'''PB''': 100 mM Tris Acetate, 4.5 M guanidine hydrochloride, 25% Isopropanol, pH 6.9 '''PE''': 10 mM Tris-HCl, 10 mM NaCl, 80% Ethanol, pH 8.0

'''Low-endotoxin Wash''': 1% n-octyl-β-D-Solution thioglucopyranoside, 1.6 M guanidine hydrochloride

'''Buffer B1''' (bacterial lysis buffer)

50 mM Tris-HCl pH 8.0 50 mM EDTA pH 8.0 0.5% Tween-20 0.5% Triton-X100 RNAse A 200 ug/l

'''Buffer B2''' (bacterial lysis buffer)

3 M Gu-HCl 20% Tween-20

'''Buffer C1''' (cell lysis buffer) (store at +4)

1.28 M sucrose 40 mM Tris-HCl pH 7.5 20 mM MgCl2 4% Triton X-100

'''Buffer G2''' (digestion buffer)

800 mM GU-HCl 30 mM Tris-HCl pH 8.0 30 mM EDTA pH 8.0 5% Tween-20 5% Triton-X100

'''Buffer Y1''' (yeast lysis buffer) (store at +4)

1 M Sorbitol 100 mM EDTA pH 8.0 14 mM beta mercaptoethanol (added just before use)

'''Buffer PAA '''(PAGE gel elution of DNA)

500 mM NH4Ac 100 mM MgAc2 1 mM EDTA 0.1% SDS

'''Buffer ENDO-1'''

50 mM MOPS or HEPES (pH 7.00 with NaOH) 750 mM sodium chloride 10% (w/v) Triton X-114 20% (v/v) isopropanol

 '''Buffer W5-ENDO'''

100 mM sodium acetate (pH 5.0 with acetic acid) 750 mM sodium chloride 1% (w/v) Triton X-114

'''Buffer W7-ENDO, Qiagen ER buffer'''

50 mM MOPS (pH 7.0 with NaOH) 750 mM sodium chloride 10% (w/v) Triton X-114 or 10% (w/v) Triton X-100 or 20% NP 40

10% ethanol

Add to cleared lysate at 10:1 ratio, e.g., add 3 ml to 30 ml cleared lysate.

 '''Stock Solutions:''' '''0.5 M MOPS pH 7.0''' 209.27 g MOPS (use the free acid form that USB sells) ~750 ml dH20 pH to 7.0 with 10 N NaOH make up to 2 liters with dH20 '''3M NaCl''' 350.6 g NaCl make up to 2 liters with dH20

'''1 M Tris pH8.5''' 121.1 g Tris base ~750 ml dH20 pH to 8.5 with concentrated HCl (takes ~20 ml)

'''PCR purification buffer'''

'''QXB''': 5 M GHCl

'''PB''': 5 M GuHCl, 30% isopropanol 20mM Tris-HCl pH 6.6 (25oC)

'''Buffer QG 1 '''(neutralization and binding buffer) 5.5M guanidine thiocyanate (GuSCN) 20mM Tris-HCl pH 6.6 (25oC)

'''Recipe 2''' 90.8 g NaI 1.5 g Na2SO3 100 ml of water. Filter through 0.45 um filter, then add another 0.5 g Na2SO3. Store in the coldroom in a dark bottle.

'''Recipe 3''' 6M Guanidine thiocyanate, Molecular Weight: 118.16, 71 g 50mM Tris-HCl pH 7.5 20mM EDTA pH 8.0

water to 100 ml

'''Recipe 4''' 60% Guanidine thiocyanate (5.36M), 60 g 140mM MES (2-[N-Morpholino] ethanesulfonic acid) 0.006% Phenol Red

H2O to 100 ml

0.006% Phenol Red Use 3 Volume of buffer to 1 gel volume.

Reference: <a href="[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750]" alt="[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750]" title="[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750]">[http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007750 http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007750]</a> Phenol Red is only a pH indicator. It changes color from yellow to red over the pH range 6.8 to 8.4.

7/3/2013

<img src="/eln/images/3/39/Gel_extraction_buffer_comparison.JPG" _fck_mw_filename="Gel extraction buffer comparison.JPG" _fck_mw_origimgwidth="159" _fck_mw_origimgheight="289" alt="RTENOTITLE" title="RTENOTITLE" style="vertical-align:middle;" /> H: Homemade gel extraction buffer

Q: Qiagen gel extraction buffer QG.

 '''Genomic DNA buffer'''

'''Chaotreopic Solution''': 4 M GTC, 25 mM sodium citrate, pH 7.5, 2% beta mecaptoethanol, with optional detergent (5%) NP40, TX100 etc.

'''Washing Buffer''': 10 mM Tris_HCl, pH 7.5, 80% ethanol.

'''Buffers for NucleoBond AX 500 anion exchange chromatography column (Macherey-Nagel).'''

 '''Resuspension buffer S1''': 50 mM Tris–HCl, 10 mM EDTA, 100 g/mL RNase A (Macherey-Nagel), pH 8.0.

 '''Lysis buffer S2''': 200 mM NaOH, 1% SDS.

 '''Neutralization buffer S3''': 2.8 M potassium acetate, adjusted to pH 5.1 with acetic acid.

 '''Equilibration buffer N2''': 100 mM Tris–HCl, 15% ethanol, 900 mM KCl, 0.15% Triton X-100, adjusted to pH 6.3 with H3PO4.

 '''Wash buffer N3''': 100 mM Tris–HCl, 15% ethanol, 1.15 M KCl, adjusted to pH 6.3 with H3PO4.

 '''Elution buffer N5''': 100 mM Tris–HCl, 15% ethanol, 1 M KCl, adjusted to pH 8.5 with H3PO4.

'''N2-EF''': 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 900 mM KCl, 0.5% Triton X-114.

'''N3-EF wash buffer:''' 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl, 0.5% Triton X-114

ethanol: to keep Triton X-114 in solution, not turbid.

'''N4-EF''': I think it is N3-EF without Triton-114: 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl,

'''N5-EF''' Elution Buffer: 100 mM TRIS/phosphate, pH 7.0, 15% ethanol, 1500 mM KCl.

'''S3''': 2.8 M potassium acetate, pH 5.2; stored at 4 oC. '''N2''': 900 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol. '''N3''': 1150 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol. '''N4''': 1000 mM KCl in 100 mM Tris-H3PO3, pH 6.3, 15% (v/v) ethanol.

=== Buffers for epochbiolabs.com maxiprep columns ===

VP1: 50mM Tris-HCl, pH 8.0, 10mM EDTA, With 100ug/mL RNase A; 10 ml per maxiprep

VP2: 0.2N NaOH, 1% SDS; 10 ml per maxiprep; leave at RT for 5 min to digest RNA VP3: 3M KoAc, pH 5.5

Shake column with 5 ml 98% ethanol, equilibrate column with 20 ml VPN, load supernatnat from lysate. VPN: 0.1M NaoAc, pH 5.1, 1.2M NaCl; Wash column with 30 ml VPN

VPE: 0.1M Tris-HCl, pH8.5, 1.35M NaCl, 15% Isopropanol; Elute DNA,  10 ml.

Precipitate DNA using 7.5 ml isopropanol, spin 30 min at 12,000 rpm. Wash pellet with 70% ethanol.

=== Buffer for Hangzhou silica Membrane column ===

Maxiprep:

'''Equilibration Buffer:''' 5~ 10%  NaOH.

'''Neutralization Solution (N3)''': 2M Potassium Acetate, pH 5.5

'''12 ml Binding Solution''' ('''BS'''): 5% n-octyl-β-D-thioglucopyranoside, '''8M''' guanidine hydrochloride '''12 ml Wash Solution 1 (W1):''' 100 mM Tris Acetate, 4.5 M guanidine hydrochloride, 25% Isopropanol, pH 6.9 '''12 ml Wash Solution 2''' '''(W2)''': 10 mM Tris-HCl, 10 mM NaCl, 80% Ethanol, pH 8.0

'''ETR:''' 20% Triton X-114 in isopropanol.

Other detegents:

1.5% BRIJ 35, 2.5% TWEEN 20, 1.0% TRITON X-100, 2.5% TRITON X-114

150 ml culture: 12 ml of P1, P2, N3; then add 8 ml BS; Load to column, wash with 12 ml BS, 12 ml W1, 12 ml W2. ELute with 3 ml TE (Spin).

'''Buffer AE''' (elution buffer for genomic DNA preps)

10 mM Tris-HCl 0.5 mM EDTA pH 9.0

'''Buffer P1'''

50 mM Tris-HCl pH 8.0 10 mM EDTA 100 μg/ml RNaseA

The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).

'''Buffer P2'''

200 mM NaOH 1% SDS

'''Buffer P3''' (not for spin columns, but for Qiatips, midi, maxi, giga kits)

3.0 M potassium acetate pH 5.5

'''Buffer DP3''' (for Qiagen Directprep 96-well miniprep)

3.0 M ammonium acetate pH 5.5

'''Buffer N3'''

4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8

'''Buffer PB'''

5 M Gu-HCl 30% isopropanol

'''Buffer QG'''

5.5 M guanidine thiocyanate (GuSCN) 20 mM Tris HCl pH 6.6

'''Buffer PE'''

10 mM Tris-HCl pH 7.5 80% ethanol

'''Buffer QX1 '''(for solution and binding of agarose gels)

7 M NaPO4 10 mM NaAc pH 5.3

'''Buffer QXB '''(for binding of large >3000 bp fragments to columns)

5 M GuHCl

'''Buffer QBT''' (equilibration buffer)

750 mM NaCl 50 mM MOPS pH 7.0 15% isopropanol 0.15% triton X-100

'''Buffer QC''' (wash buffer)

1.0M NaCl 50 mM MOPS pH 7.0 15% isopropanol

'''Buffer QF '''(elution buffer)

1.25M NaCl 50 mM Tris-HCl pH 8.5 15% isopropanol

'''Buffer QN'''

1.6M NaCl 50 mM MOPS pH 7.0 15% isopropanol

'''Buffer FWB2'''

1M potassium acetate, pH 5.0

'''Buffer B1 '''(bacterial lysis buffer)

50 mM Tris-HCl pH 8.0 50 mM EDTA pH 8.0 0.5% Tween-20 0.5% Triton-X100 RNAse A 200 μg/l

'''Buffer B2''' (bacterial lysis buffer)

3 M Gu-HCl 20% Tween-20

'''Buffer C1''' (cell lysis buffer) (store at +4)

1.28 M sucrose 40 mM Tris-HCl pH 7.5 20 mM MgCl2 4% Triton X-100

'''Buffer G2''' (digestion buffer)

800 mM GU-HCl 30 mM Tris-HCl pH 8.0 30 mM EDTA pH 8.0 5% Tween-20 5% Triton-X100

'''Buffer Y1''' (yeast lysis buffer) (store at +4)

1 M Sorbitol 100 mM EDTA pH 8.0 14 mM beta mercaptoethanol (added just before use)

'''Buffer PAA''' (PAGE gel elution of DNA)

500 mM NH4Ac 100 mM MgAc2 1 mM EDTA 0.1% SDS

'''Column Regeneration'''

The blue and purple Qiagen columns are identical in formulation. They are interchangeable and can be reused multiple times after treatment with hydrochloric acid, re-equilibration, and wash. Very likely this protocol can be used with other similar columns.

The reuse protocol is:

Save the collection tubes and columns after elution of DNA Fill the column with 700 μl of 1 M HCl Cap and store in an airtight container for at least 24 hours (less than a month) Wash the columns and collection tubes in a large beaker of water Assemble the column and collection tube and wash the column with 700 μl of DI water, discarding the water Repeat Fill the column with 700 μl of buffer QBT and spin down, discarding the buffer Place the columns in an airtight plastic bag for storage Wash the collection tubes, air dry, and store them for reuse

1) When you are finished with the gel purification from Qiagen, place the used columns into the paired, used collection tubes.

2) Fill used column with 700 mL of 1 M HCl.

3) Store with cap closed in an airtight container for at least 24 hours (up to 30 days).

4) Once you have a bunch of columns and collection tubes in the 1 M HCl, to prepare a column for use:

Thoroughly rinse the columns and collection tubes in a large beaker of dH2O.

Shake/ wipe off excess water. Place each column into a collection tube. Fill each column with 700 uL sterile dH2O. Spin at maximum speed in a tabletop centrifuge for 1 minute. Discard the water.

Repeat the previous step a second time.

Equilibrate the column by filling it with 700 µL Buffer QBT. Spin at maximum speed in a tabletop centrifuge for 1 minute.

Discard the buffer. Spin again for 1 minute to remove excess QBT buffer from the column.

Put clean columns into a plastic bag. Rinse the collection tubes in a large beaker of dH2O.

Pour off water and allow tubes to air dry. Place into a plastic bag. The columns are now ready for use.

Note: After 9 rounds of regeneration, columns have shown optimal performance in a series of assays (Siddappa et al. 2007).

1M HCl

FW HCl = 36.47; 37% HCl = 370 g/L » 10 M

Note: for HCl, 1 Molar = 1 Normal

'''Qiagen Buffer QBT '''(Equilibration Buffer)

[750 mM NaCl, 50 mM MOPS (pH 7.0),15% ethanol, 0.15% Triton X-100]

Dissolve 43.83g NaCl, 10.46g MOPS (free acid) in 800 mL dH2O. Adjust pH to 7.0. Add 150 mL 100% ethanol and 15 mL 10% triton X-100 solution. Adjust volume to 1 liter.

Store at room temperature. Invitrogen Buffers ==

'''RNase A''': 20 mg/mL in Resuspension Buffer (R3) '''Lysis Buffer (L7)''': 0.2 M NaOH 1% (w/v) SDS '''Precipitation Buffer (N3)''': 3.1 M Potassium acetate, pH 5.5 '''Equilibration Buffer (EQ1)''': 0.1 M Sodium acetate, pH 5.0, 0.6 M NaCl, 0.15% (v/v); Triton X-100 '''Wash Buffer (W8)''': 0.1 M Sodium acetate, pH 5.0, 825 mM NaCl '''Elution Buffer (E4)''': 100 mM Tris-HCl, pH 8.5, 1.25 M NaCl '''TE Buffer (TE)''': 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA

== Nucleobond DNA Maxi-Prep Buffers ==

Soln P1 or S1: TE Resuspension Buffer (Store at 4 °C)

50 mM Tris pH 8.0

10 mM EDTA (8.0) 0.1 mg/mL (100ug/ml) RNAse A

'''Soln P2 or S2''': NaOH/SDS Lysis Buffer (Store at RT)

0.2 M NaOH 1% SDS

'''Soln P3 or S3''': KOAC Neutralization Buffer (Store at 4 °C)

2.8 M KOAc Glacial Acetic Acid: added until pH is 5.1

Soln Nucleobond '''N2''': Equilibration Buffer (Store at RT)

100 mM Tris 15% EtOH 900 mM KCl 0.15% Triton X-100 Adjust with Phosphoric Acid to pH  6.3

Soln Nucleobond '''N3''': Wash Buffer (Store at RT)

100 mM Tris 15% EtOH 1.15 M KCl Phosphoric Acid: added until pH is 6.3

'''ENDO-EF''':

It has ethanol and Triton X-100 or Triton X-114.

100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl, 0.5% Triton X-114

'''N5-EF'''

100 mM TRIS/phosphate, pH 6.8~7.0, or 0.1~0.2 M Phosphate Buffer, pH 6.8~7.0, 1.8 to 2 M NaCl (or 1.8 M KCl).

Soln Nucleobond '''N5''': Elution Buffer (Store at RT)

100 mM Tris 15% EtOH 1 M KCl Phosphoric Acid: added until pH is 8.5

{|
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | Buffer Name 
| bgcolor="#ffffcc" | Recipe 
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe 
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" |   N2
| bgcolor="#ffffcc" | 1 L
| bgcolor="#ffffcc" | N3
| bgcolor="#ffffcc" | 1 L
| bgcolor="#ffffcc" | N4 
| bgcolor="#ffffcc" | 1L 
| bgcolor="#ffffcc" | N5
| bgcolor="#ffffcc" | 1L
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 12.1 g
| bgcolor="#ffffcc" | 100 mM Tris
| bgcolor="#ffffcc" | 12.1 g
| bgcolor="#ffffcc" | 100 mM Tris 
| bgcolor="#ffffcc" | 12.1 g 
| bgcolor="#ffffcc" | 100 mM Tris
| bgcolor="#ffffcc" | 12.1 g
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 15 % EtOH
| bgcolor="#ffffcc" | 150 ml
| bgcolor="#ffffcc" | 15 % EtOH
| bgcolor="#ffffcc" | 150 ml
| bgcolor="#ffffcc" | 15 % EtOH 
| bgcolor="#ffffcc" | 150 ml 
| bgcolor="#ffffcc" | 15 % EtOH
| bgcolor="#ffffcc" | 150 ml
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 900 mM KCl
| bgcolor="#ffffcc" | 67.1 g
| bgcolor="#ffffcc" | 1.15 M KCl
| bgcolor="#ffffcc" | 85.7 g
| bgcolor="#ffffcc" | 1.15 M KCl 
| bgcolor="#ffffcc" | 85.73 g 
| bgcolor="#ffffcc" | 1 M KCl
| bgcolor="#ffffcc" | 74.56 g
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 0.15 % TX 100
| bgcolor="#ffffcc" | 1.5 ml
| bgcolor="#ffffcc" | 0.15 % TX 100
| bgcolor="#ffffcc" | 1.5 ml
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''8.5''' with H3PO4 
|}

N2 900 mMKCl 100 mM Tri pH6.3 15% Ethanol 0.5% Triton X-114

N3 1150 mM KCl 100 mM Tris pH 6.3 15% Ethanol 0.5% Triton X-114

N4 1150 mM KCl 100 mM Tris pH 6.3 15 Ethanol

N5 1.5M KCl 100 mM Tris pH 7.0 15% Ethanol

'''N and L Series Buffer Preparation for Normal Processing of Nucleobond AX Cartridges'''

The most important factors when making up the N or L series of buffers for Nucleobond AX cartridges are the concentration of KCl and the pH. Even slight deviations in these could change the elution profiles of different nucleic acids. The following guidelines are presented primarily for those laboratories who use Nucleobond AX cartridges with buffers that they choose to make themselves. The protocol assumes that the buffers and cartridges will be used for both DNA and RNA purifications. If only DNA is to be purified, the DEPC treatment in step 1 can be omitted. The following procedure will make one liter of each buffer.

Prepare DEPC (diethylpyrocarbonate)-treated water by adding 0.1 ml DEPC per 100 ml distilled water. Stir for at least two hours and autoclave for 30-40 minutes to obtain RNAse-free sterile water.

 For each N or L solution, begin by adding 12.11 g of Tris base to 600 ml of water and stir to dissolve. Add KCl to the required final concentration for each buffer as follows:

N1 or L1:  29.82 g KCl N2 or L2:  67.10 g KCl L3: 74.55 g KCl N3:  85.73 g KCl N4 96.92 g KCl N5 or L5 74.55 g KCl

Stir to dissolve and autoclave. Allow the autoclaved buffers to cool to room temperature and add 150 ml of absolute (100%) ethanol to each. (See NOTE) Adjust the pH of each buffer with phosphoric acid as follows:

N1 or L1 to 6.3 N2 or L2 to 6.3 N3 or L3 to 6.3 N4 to 6.3 N5 or L5 to 8.5

(Be very careful not to "overshoot" the appropriate pH for each solution when titrating because correcting it with a basic solution will alter the salt concentration of the buffer which, in turn, could alter cartridge performance.)

 For each buffer add enough autoclaved, distilled water (DEPC-treated, if required) to reach a final volume of 1000 ml. Store at room temperature in a tightly closed container to prevent evaporation.

NOTE 1: The ethanol and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the ethanol or acid approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.

'''N5 and L5 Elution Buffer Preparation for High GC Content DNA'''

DNA with GC contents higher than 60% (or with large regions of high GC content) may continue to bind a Nucleobond AX cartridge even under normal elution conditions. In such cases, some type of DNA denaturant must be added in order to do the job. The final buffer composition that has been shown to work for the modified N5 and L5 elution buffers is as follows: 50% formamide, 1.0 M KCl, 15% EtOH, 100 mM Tris-phosphate, pH 8.5. The following procedure will make one liter of this buffer.

Begin by adding 12.11g of Tris base to 250 ml of deionized water and stir to dissolve. Add 74.55g of KCl to the bring it to the required final concentration of 1.0M. Stir to dissolve and autoclave.

 Allow the autoclaved buffer to cool to room temperature and add 150 ml of absolute (100%) ethanol1 and 500 ml of deionized formamide. (The formamide can be deionized by treating it with a mixed bed resin to bring the pH close to 7.)

 Adjust the pH to 8.5 with phosphoric acid.

 NOTE: The ethanol, formamide, and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the reagent approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.

(Be very careful not to "overshoot" the appropriate pH for each solution when titrating because correcting it with a basic solution will alter the salt concentration of the buffer which, in turn, could alter cartridge performance.) Add enough autoclaved, distilled water to reach a final volume of 1000 ml. Store at room temperature in a tightly closed container to prevent evaporation.

Formulas for Qiagen Kit Buffers

Do not autoclave solutions containing ethanol, isopropanol or MOPS; use sterile filtration if necessary. Buffer AE (elution buffer for genomic DNA preps) 10 mM Tris-HCl 0.5 mM EDTA pH 9.0 Buffer P1 50 mM Tris-HCl pH 8.0 10 mM EDTA 100 μg/ml RNaseA The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101). Buffer P2 200 mM NaOH 1% SDS Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits) 3.0 M potassium acetate pH 5.5 Buffer DP3 (for Qiagen Directprep 96-well miniprep) 3.0 M ammonium acetate pH 5.5 Buffer N3 4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8 Buffer PB 5 M Gu-HCl 30% isopropanol Buffer QG 5.5 M guanidine thiocyanate (GuSCN) 20 mM Tris HCl pH 6.6 Buffer PE 10 mM Tris-HCl pH 7.5 80% ethanol Buffer QX1 (for solution and binding of agarose gels) 7 M NaPO4 10 mM NaAc pH 5.3 Buffer QXB (for binding of large >3000 bp fragments to columns) 5 M GuHCl Buffer QBT (equilibration buffer) 750 mM NaCl 50 mM MOPS pH 7.0 15% isopropanol 0.15% triton X-100 Buffer QC (wash buffer) 1.0M NaCl 50 mM MOPS pH 7.0 15% isopropanol Buffer QF (elution buffer) 1.25M NaCl 50 mM Tris-HCl pH 8.5 15% isopropanol Buffer QN 1.6M NaCl 50 mM MOPS pH 7.0 15% isopropanol Buffer FWB2 1M potassium acetate, pH 5.0 Buffer B1 (bacterial lysis buffer) 50 mM Tris-HCl pH 8.0 50 mM EDTA pH 8.0 0.5% Tween-20 0.5% Triton-X100 RNAse A 200 μg/l Buffer B2 (bacterial lysis buffer) 3 M Gu-HCl 20% Tween-20 Buffer C1 (cell lysis buffer) (store at +4) 1.28 M sucrose 40 mM Tris-HCl pH 7.5 20 mM MgCl2 4% Triton X-100 Buffer G2 (digestion buffer) 800 mM GU-HCl 30 mM Tris-HCl pH 8.0 30 mM EDTA pH 8.0 5% Tween-20 5% Triton-X100 Buffer Y1 (yeast lysis buffer) (store at +4) 1 M Sorbitol 100 mM EDTA pH 8.0 14 mM beta mercaptoethanol (added just before use) Buffer PAA (PAGE gel elution of DNA) 500 mM NH4Ac 100 mM MgAc2 1 mM EDTA 0.1% SDS Buffer PNI (purification of oligonucleotides 17 nt and greater) 40% (v/v) 5 M guanidinium chloride 60% (v/v) isopropanol  Concentration of guanidinium determined by drying down supplied concentrate and measuring mass. Concentration of isopropanol determined by calculation from the supplied user manual. 

(Source: [1], US Patent 6,383,393) The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3
[[Category:Buffer Recipe]]

File:2951 bglII.JPG

2022-01-22T02:56:43Z

Paul.xu:

File:2951 EagI digestion.JPG

2022-01-17T01:30:51Z

Paul.xu:

MRNA Production

2022-01-16T21:43:35Z

Paul.xu: Created page with "{| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | Name | ID | Location | Size | Link | Digestion |- | T7-VEE-TagGFP2-E3L | 2794 | 35A6 | 11047..."

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
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| Name
| ID
| Location
| Size
| Link
| Digestion
|-
| T7-VEE-TagGFP2-E3L 
| 2794 
| 35A6
| 11047
| 
| Xba1
|-
| T7ag-VEE-eCas9 
| 2947 
| 35H5
| 4831
| 
| Xba1
|-
| 
| 
| 
| 
| 
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[[Category:MRNA]]

Proteins in Freezer

2021-12-02T23:24:40Z

Paul.xu:

== Box P1 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified by
|-
| 
| 
| Taq
| 
| 
| 1.19mg/ml
| 
| 3
| 
| 
| 
|-
| 
| 
| Taq 
| 
| 
| 1.42 mg/ml
| 
| 3
| 
| 
| 
|-
| 
| 
| Taq 
| 
| 
| 4.21 mg/ml
| 
| 4
| 
| 
| 
|-
| 
| 
| Taq 
| 
| 
| 0.48 mg/ml
| 
| 2
| 
| 
| 
|-
| 67-2038
| 
| Taq 
| 
| 
| 1.5mg/ml
| 
| 2
| 
| 
| 
|-
| 20130724
| 
| KOD 
| 
| 
| 10mg/ml
| 0.5 ml
| 2
| 
| 
| 
|-
| 
| 
| Phusion
| 6-27-12
| CM
| 4.4 mg/ml
| 
| 16
| 
| 
| 
|-
| 
| 
| Phusion 
| 
| 
| 17.5 mg/ml
| 
| 
| 
| 
| 
|-
| 
| 
| Phusion 
| 5/18/18
| QC
| 
| 
| 
| 
| 
| 
|-
| 20140203
| 
| Kfu
| 
| 
| 1mg/ml
| 
| 
| 
| 
| 
|-
| 
| 
| Kfu 
| 
| 
| 1.26mg/ml 
| 
| 
| 
| 
| 
|-
| 2013/8/14 
| 1498
| Taq DNA Ligase 
| 2013/8/14 
|  pT7A3-TaqDNAligase 
| 9mg/ml
| 
| 
| 
| 
| 
|-
| 162-1483
| 
| T4 DNA ligase
| 6/26/19 
| 
| 2.8 mg/ml 
| 
| 
| 
| 
| 
|}

== Box P2 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| 
| 1747
| MMLV
| 2013/10/11
| pT7A30-MMLV
| 6mg/ml
| 
| 2
| 
| 
| 
|-
| 81-2264
| 
| Gst-Polymerase
| 
| 
| 29.6 mg/ml
| 
| 6
| 
| 
| 
|-
| 
| 1811
| V-Pol
| 2013/10/15
| 
| 44.6 mg/ml
| 
| 2
| 
| 
| 
|-
| 
| 
| V-Pol
| 2013/10/15 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 2462
| TN5 Transposase
| 
| 
| 8.86 mg/ml 
| 
| 5
| 
| 
| 
|-
| 
| 
| TN5 Transposase 
| 
| 
| 
| 10 ml
| 1
| 
| 
| 
|-
| 
| 
| LacI-SV40
| 2014/2/11
| 
| 4.75 mg/ml
| 
| 
| 
| 
| 
|-
| 
| 1797
| 3C-MBP
| 2013/8/14
| pCold-6His-3C
| 2.6mg/ml
| 
| 6
| 
| 
| 
|-
| 1402/11
| 1941
| 
| 
| 
| 0.45ml
| 
| 
| 
| 
| 
|-
| 
| 
| FN3K
| 20131014
| 
| 0.93mg/ml
| 
| 
| 
| 
| 
|-
| 168-2565 
| 2565
| Thermostable MMLV
| 7/18/19
| 
| 0.46mg/ml
| 6ml
| 3 vials
| 
| 
| 
|-
| 
| 
| MMLV EG-1029
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P3 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| 82-2392
| 
| UVSX
| 
| 
| 18.6 mg/ml
| 500ul
| 24
| 
| 
| 
|-
| 72-2196
|
2196

| EG-1063 PBCV-1 DNA Ligase Chorella virus DNA Ligase 
| 10/8/14 
| 8mg/ml
| 
| 
| 14 
| 
| 
|-
| 20130806
| 
| MBP-(3C-TEV)-PKARIIB 
| 
| 
| 
| 
| 6
| 
| 
| 
|-
| 219-1593 
| 
| E coli Endonuclease IV (Endo IV), NfoEG-160 
| 6/1/21
| 
| 1mg/ml 
| 5000U
| 7
| 
| 
| Paul
|}

== Box P4 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| 20121223
| 
| T4 DNA ligase
| 
| 
| 12mg/ml
| 
| 3
| 
| 
| 
|-
| 
| 
| T4 DNA ligase 
| 
| 
| 41/mg/ml
| 
| 2
| 
| 
| 
|-
| 20130204
| 
| T3 DNA ligase 
| 
| 
| 
| 
| 15
| 
| 
| 
|-
| 20121207
| 1483
| T7 DNA ligase 
| 
| 
| 4.5mg/ml
| 
| 15
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P5 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| 58-2107
| EG-25
| Taq Muts
| 
| 
| 4mg/ml
| 
| 38
| 
| 
| 
|-
| 58-2107 
| EG-41
| Taq Muts 
| 
| 
| 4mg/ml
| 100ug
| 
| 
| 
| 
|-
| 207-1078 
| EG-39
| IHF
| 11/23/2020
| 
| 3mg/ml
| 90 ul
| 
| 
| 
| 
|-
| 206-1074 
| EG-39 
| IHF
| 11/23/2020
| 
| 2mg/ml 
| 90 ul 
| 
| 
| 
| 
|-
| 208-1203 
| EG-39
| GST-IHF
| 11/23/2020 
| 
| 3mg/ml 
| 90 ul 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

{| border="1" cellspacing="1" cellpadding="1" style="width: 800px;"
|-
| 90 ul
|}

== Box P6 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Cat
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  
|
| His-MBP-Muts
| EG-197 
| 10/28/11
| all tubes 50% Gly
| 1.5mg/ml
| 0.5mg
| 41
|
|
|
|-
| 0117
| EG-9
| Taq-Muts-protein
| 
|
|
|  12mg/ml
| 500ug
| 11
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P7 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  
|
| Taq-Muts
|
| 20%Gly
| 1mg/ml
| 50ug
| 20
|
|
|
|-
| 88-212
| 212
| Tth-Muts
| 7/24/15
|
| 2.52mg/ml
|
| 48
| 
| 
| 
|-
| 158-212
| 212
| Tth-Muts
| 3/8/19
| P7R2.4 is good, red cap 
| 2.38
| 50ug
| 48 +1 .5ml
| 
| 
| 
|-
| 220-129 
| 129 
| Tth-Muts 
| Jul 03 2021 
| Ni--> QFF QFF: large column 2.5 cmX10 cm QFF, dilute Buffer B ( 500 mM NaCl, 25 mM Tris-HCl, pH 8.0, 150 mM Imadazole) eluante with 2 vol of dilution buffer (25 mM Tris-HCl, pH8.0, 50 mM NaCl), load to the column,  wash column with dilution buffer, then elute with 1 M NaCl in Dilution buffer. Small column (1.6 cmX10 cm), dilute  eluant from the large QFF column further with 1 vol of dilution buffer, load to small column,  wash column with dilution buffer, then elute with 1 M NaCl in Dilution buffer.  
|
Large column:  2mg/ml, 6 X 1 ml, total 12 mg 

Small column: 2mg/ml, 8.5 ml, total 17 mg

| 
| 
| 
| 
| 
|}

== Box P8 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  
|
| Taq
| 04/21/2016
|
|
|
| 79
|
|
|
|-
|
|
|
|
|
|  
|
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P9 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  
|
| Taq-Muts
|
| green cap
| 8.6mg/ml
| 0.5mg
| 17
|
|
|
|-
|
|
|
|
| no name, probably is Tth Muts protein6Xhis-Tth-Muts
|  7.4mg/ml
| 500ug
| 32
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P10 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| 89-1529
|
| Ecoli Rec A
| 7/27/15
|
| 1.6mg/ml
| 1mg
| 16
|
|
|
|-
| 89-1529
|
| Ecoli Rec A
| 7/27/15
|
|  1.6mg/ml
| 200ug
| 18
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P11 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  20130224
|
| GST-3C
|
|
| 6.78mg/ml
|
| 13
|
|
|
|-
|
|
| TEV
| 02/03/11
|
|  1mg.ml
|
| 4
| 
| 
| 
|-
| 
| 
| GST-TEV
| 
| 
| 
| 
| 2
| 
| 
| 
|-
| 12152110
| RP-8
| Turbo-TEV protesse
| 
| 
| 10U/ul 1mg/ml
| 
| 38
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P12 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  
|
| Taq Ligase
|
|
| 2mg/ml,20000 unit
|
|
|
|
|
|-
|
|
| Tag Ligase 
|
|
| 3.77mg/ml
|
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P13 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified by
|-
| 
| 
| unknown, maybe transfection reagent
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P14 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| 57-1005,155-1005
| EG-1068
| Tat-GFP
|
|
|
| 1mg
| 17
|
|
|
|-
| 56-375
| RP-7
| Cre recombinase
|
|
| 2mg/ml
| 1mg
| 9
| 
| 
| 
|-
| 20130826
| RP-7
| Cre recombinase
| 
| 
| 5mg/ml
| 1mg
| 4
| 
| 
| 
|-
| 121913
| EG-1001
| HNC-Cre recombinase
| 
| 
| 2mg/ml
| 1mg
| 11
| 
| 
| 
|-
| 
| RP-18
| HTN-GFP
| 
| 
| 1mg/ml
| 
| 1
| 
| 
| 
|-
| 5414
| RP-22
| FLP recombinase
| 
| 
| 1mg/ml
| 0.5mg
| 
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P15 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Lot
| ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
|  63-2288
| 2288
| SUMO protein SMT3
|
|
| 4mg/ml
| 0.5mg
| 3
|
|
|
|-
|
| 2289
| SMT3
|
|
| 1mg/ml
|
| 3
| 
| 
| 
|-
| 70-212
| 212
| Tth-Muts
| 
| 
| 5.8mg/ml
| 
| 6
| 
| 
| 
|-
| 
| 211
| MBP-Taq-Muts
| 
| 
| 14mg/ml
| 
| 2
| 
| 
| 
|-
| 
| 
| MBP--Taq-Muts
| 
| 
| 3.5mg/ml
| 
| 1
| 
| 
| 
|-
| 
| 
|
429--pHisMBP-TVMV,

1512-->Phi29 DNA Polymerase,???

| 
| 
| 1.47mg/ml
| 
| 3
| 
| 
| 
|-
| 1-165
| 
| M1wt
| 
| 
| 
| 
| 2
| 
| 
| 
|-
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

== Box P16 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| style="width: 54px;" | Lot
| style="width: 7px;" | ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| style="width: 54px;" |  
| style="width: 7px;" | 1737
| TRX NN
|
|
| 4mg/ml
|
| 9
|
|
|
|-
| style="width: 54px;" | 65-1737
| style="width: 7px;" | 1737
| TRX
|
|
| 13mg/ml
| 0.5mg
| 4
| 
| 
| 
|-
| style="width: 54px;" | 
| style="width: 7px;" | 1736
| NusA
| 
| 
| 4mg/ml
| 
| 13
| 
| 
| 
|-
| style="width: 54px;" | 
| style="width: 7px;" | 1736
| NusA
| 
| 
| 4.5mg/ml
| 
| 7
| 
| 
| 
|-
| style="width: 54px;" | 
| style="width: 7px;" | 1736
| NusA
| 
| 
| 3mg/ml
| 
| 15
| 
| 
| 
|-
| style="width: 54px;" | 170-2616 
| style="width: 7px;" | 2616
| CYFRA 21-1 
| 
| 
| 2mg/ml
| 30 mg
| 11
| 
| 
| 
|-
| style="width: 54px;" | 171-1185 
| style="width: 7px;" | 1185
| MBP
| 
| 
| 2mg/ml
| 14 mg
| 4
| 
| 
| 
|}

== Box P17 ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| style="width: 54px;" | Lot
| style="width: 7px;" | ID
| Name
| Date
| Description
| Concentration
| Size
| Quantity
| Position
| QC
| Purified By
|-
| style="width: 54px;" |  182-2565
| style="width: 7px;" | 2565
| Thermostable reverse transcriptases MMLV
| 6/22/2020
|
|
13mg/ml white cap

1.3 mg/ml blue cap

|
21

10

|
|
|
|
|-
| style="width: 54px;" | 184-2779 
| style="width: 7px;" | 2779
| Polynucleotide phosphorylase 
| 7/1/2020
| 
|
7mg/ml.

1 mg/ml

|
3

1

| 
| 
| 
| 
|-
| style="width: 54px;" | 216-2932 
| style="width: 7px;" | 
| Cas13A (sumo digested) 
| 2/8/2021
| 
| 16.72 mg/ml 
| 4
| 
| 
| 
| 
|-
| style="width: 54px;" | 221-2565
| style="width: 7px;" | 
| MMLV Thermostable, EG-1061
| 12/2/2021
| 
| 5 mg/ml
| 15 ml
| 
| 
| 
| 
|-
| style="width: 54px;" | 217-2932 
| style="width: 7px;" | 
| Cas13A (not digested) 
| 2/16/2021 
| 
|
5.13mg/ml 

2.5 mg/ml

|
11

1

| 
| 
| 
| 
|}
[[Category:Inventories]]