ELN - User contributions [en]

File:2616 Q.png

2019-10-04T21:48:00Z

Crm:

File:2616 Ni.png

2019-10-04T21:46:14Z

Crm:

Multitag iFlp Expression Screening

2019-09-26T22:37:24Z

Crm:

== Protein Expression Screening ==

Cells: Nico21(DE3)pLysN, 15 ml LB/KC, with 1 X5052

{| border="1" cellspacing="0"
|-
| #
| Clone ID
| Name
| Induction
| His Tag
| pI MW
| style="width: 69px;" | Expression
| style="width: 53px;" | Anomaly observation
|-
| 1
| 2596
| pT7A30-D7324-6H-CCMV
| 1 mM IPTG, 18 oC o/n
| 
| 9.89 / 23.4
| style="width: 69px;" | 
| style="width: 53px;" | 
|-
| 2
| 2586
| pET28a-6H-D7324-AP205
| 1 mM IPTG, 18 oC o/n 
| 
| 9.88 / 17.6
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed
|-
| 3
| 2595
| pDB.His.GST-D7324
| 1 mM IPTG, 18 oC o/n 
| 
| 6.90 / 31
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 4
| 2594
| pDB.His.GST-SpyCatcher
| 1 mM IPTG, 18 oC o/n 
| 
| 5.75 / 39.3
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 5
| 2593
| pDB.His.GST-SpyCatcher-AP205
| 1 mM IPTG, 18 oC o/n 
| 
| 5.99 / 53.6
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 6
| 2585
| pT7A3-TRX-multi-Tags
| 1 mM IPTG, 18 oC 4h 
| 
| 4.67 / 49.7
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 7
| 2587
| pT7A3-GST-multi-Tags
| 1 mM IPTG, 18 oC o/n 
| 
| 4.97 / 63.9
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 8
| 2604
| pCold-MBP-multitag
| 1 mM IPTG, 18 oC o/n 
| 
| 4.88 / 80.6
| style="width: 69px;" | 
| style="width: 53px;" |  
|-
| 9
| 2580
| pET28a-Qbeta-Tat-6His
| 1 mM IPTG, 18 oC o/n 
| 
| 10.35 / 18.7
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 10
| 2579
| pET28a-6H-Tat-AP205
| 1 mM IPTG, 18 oC o/n 
| 
| 10.54 / 18.3
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed
|-
| 11
| 129
| pT7A30-TthMuts
| 1 mM IPTG, 18 oC o/n 
| 
| 5.87 / 91.8
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| style="background-color: rgb(255, 255, 153);" | 12
| style="background-color: rgb(255, 255, 153);" | 136
| style="background-color: rgb(255, 255, 153);" | pL6H-Tat-Cre
| style="background-color: rgb(255, 255, 153);" | 1 mM IPTG, 18 oC o/n 
| style="background-color: rgb(255, 255, 153);" | 
| style="background-color: rgb(255, 255, 153);" | 10.30 / 42.7
| style="width: 69px; background-color: rgb(255, 255, 153);" | 
| style="width: 53px; background-color: rgb(255, 255, 153);" | 
|-
| style="background-color: rgb(255, 255, 153);" | 13
| style="background-color: rgb(255, 255, 153);" | 135
| style="background-color: rgb(255, 255, 153);" | pQE-80L-HTNCre
| style="background-color: rgb(255, 255, 153);" | 1 mM IPTG, 18 oC o/n 
| style="background-color: rgb(255, 255, 153);" | 
| style="background-color: rgb(255, 255, 153);" | 10.30 / 42.8
| style="width: 69px; background-color: rgb(255, 255, 153);" | 
| style="width: 53px;" | 
|-
| 14
| 170
| pT7-MBP-(tev)-GST-(3c)-Tat-NLS-iFLP-6H
| 1 mM IPTG, '''18''' oc 4 hours 
| 
| 8.64 / 123.3
| style="width: 69px;" | 
| style="width: 53px;" | 
|-
| 15
| 169
| pBad-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H
| ara, 0.2% 1 mM IPTG, 18 oC o/n
| 
| 8.64 / 123.4
| style="width: 69px;" | 
| style="width: 53px;" | most cells lysed 
|-
| 16
| 164
| pT7A3-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H
| 1 mM IPTG, '''37''' oC 4h 
| 
| 8.64 / 123.4
| style="width: 69px;" | 
| style="width: 53px;" | 
|-
| 17
| 162
| pCold-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H
| 1 mM IPTG, 18 oC o/n 
| 
| 8.64 / 123.4
| style="width: 69px;" | 
| style="width: 53px;" | 
|-
| 18
| 211 
| pDBHisMBP-TaqMuts 
| 1 mM IPTG, 18 oC o/n 
| 
| 5.40 / 134 
| style="width: 69px;" | 
| style="width: 53px;" | 
|-
| 19
| 941
| pT7A3-pMBP-T7-Endo-1 
| 1 mM IPTG, '''37''' oC 4h 
| 
| 7.70 / 63.4 
| style="width: 69px;" | 
| style="width: 53px;" | 
|-
| 20
| 2619
| pT7A3-pMBP-T7EndoI-PA/A 
| 1 mM IPTG, '''37''' oC 4h 
| 
| 7.35 / 64.4 
| style="width: 69px;" | 
| style="width: 53px;" | 
|}

[[File:Protein exp screening 9 23 19.jpg|RTENOTITLE]]

S: soluble, P: inclusion body  

M: PageRuler Plus, 10, 15, 25, 55, 70,100, 130, 250

[[File:Protein exp screening 9 23 19 2.jpg|RTENOTITLE]]

9/26/19

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| ID
| Sample 
| Induction
| 
| Expression
|-
| 1
| 162
| soluble
| 1 mM IPTG, 18 oC, 4 hours
| 
| 
|-
| 2
| 162
| soluble 
| 1 mM IPTG, 18 oC, 4 hours, 3.3 mM DTT 
| 
| 
|-
| 3
| 162
| soluble 
| 1 mM IPTG, 18 oC, 4 hours, 6.6 mM DTT 
| 
| 
|-
| 4
| 162
| soluble 
| 1 mM IPTG, 18 oC, 4 hours, 10 mM DTT 
| 
| 
|-
| 5
| 164 
| soluble 
| 0.2 mM IPTG, 25 oC, 4 hours  
| 
| 
|-
| 6
| 164 
| soluble 
| 0.2 mM IPTG, 25 oC, 4 hours, 3.3 mM DTT 
| 
| 
|-
| 7
| 164 
| soluble 
| 1 mM IPTG, 25 oC, 4 hours  
| 
| 
|-
| 8
| 164 
| soluble 
| 1 mM IPTG, 25 oC, 4 hours, 3.3 mM DTT 
| 
| 
|-
| M
| 
| 
| 
| 
| 
|-
| 9
| 162 
| IB
| 1 mM IPTG, 18 oC, 4 hours 
| 
| 
|-
| 10
| 162 
| IB 
| 1 mM IPTG, 18 oC, 4 hours, 3.3 mM DTT 
| 
| 
|-
| 11
| 162 
| IB 
| 1 mM IPTG, 18 oC, 4 hours, 6.6 mM DTT 
| 
| 
|-
| 12
| 162 
| IB
| 1 mM IPTG, 18 oC, 4 hours, 10 mM DTT 
| 
| 
|-
| 13
| 164 
| IB 
| 0.2 mM IPTG, 25 oC, 4 hours, 3.3 mM DTT 
| 
| 
|-
| 14
| 164
| IB 
| 1 mM IPTG, 25 oC, 4 hours, 3.3 mM DTT 
| 
| 
|}

[[File:162 164 exp screening.jpg|RTENOTITLE]]

transferred to membrane

[[File:162 164 exp screening wb.jpg|RTENOTITLE]]

[[File:162 164 exp screening wb.jpg]][[File:162 164 exp screening wb2.jpg]]

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| sup
|}

2615 pCold-MBP-(tev)-GST-(3c)-Ac-LysargiNase

2019-09-20T16:06:55Z

Crm: Created page with "Category:CloningFile:2616 2615 cloning.JPG"

[[Category:Cloning]][[File:2616 2615 cloning.JPG]]

2616 pET28a-hFth-CYFRA 21-1-avitag-6H

2019-09-20T16:05:43Z

Crm: Created page with "File:2616 2615 cloning.JPG Category:Cloning"

[[File:2616 2615 cloning.JPG]]
[[Category:Cloning]]

2619 pT7A3-pMBP-T7EndoI-PAtoA

2019-09-20T16:04:45Z

Crm: Created page with "File:2619 PCR1.JPGFile:2619 PCR2.JPGFile:2619 Vector digestion.JPGFile:2619 digestion1.JPGFile:2619 digestion2.JPG Category:Cloning"

[[File:2619 PCR1.JPG]][[File:2619 PCR2.JPG]][[File:2619 Vector digestion.JPG]][[File:2619 digestion1.JPG]][[File:2619 digestion2.JPG]]
[[Category:Cloning]]

2585 pT7A3-TRX-multi-Tags

2019-09-07T16:30:29Z

Crm:

2585 in pJet1.2 digested with Nhe1-Xho1

2596 in pJet1.2 digested with Nde1 Xho1

[[File:2586 2596 digestion.JPG|RTENOTITLE]]

[[File:2585 digestion1.JPG|RTENOTITLE]][[File:2585 digestion2.JPG|RTENOTITLE]]

9/7

use 1833 as vector

pT7A3-TRX-multi-tags

http://excellgen.net/eln/images/d/d5/2585_digestion3.JPG[[File:2585 digestion4.JPG]]

[[Category:Cloning]]

2596 pT7A30-D7324-6H-CCMV

2019-08-31T15:11:05Z

Crm: Created page with "File:2596 digestion2.JPG Category:Cloning"

[[File:2596 digestion2.JPG]]
[[Category:Cloning]]

2594 pDB.His.GST-SpyCatcher

2019-08-28T20:01:03Z

Crm:

[[File:2593 digestion.JPG|RTENOTITLE]]

[[File:2604 2587 2594.JPG]]
[[Category:Cloning]]

2587 pT7A3-GST-multi-Tags

2019-08-28T20:00:21Z

Crm:

[[File:2604 2587 2594.JPG|RTENOTITLE]]
[[Category:Cloning]]

2604 pCold-MBP-multitag

2019-08-28T19:59:00Z

Crm: Created page with "File:2604 2587 2594.JPG Category:Cloning"

[[File:2604 2587 2594.JPG]]
[[Category:Cloning]]

2580 pET28a-Qbeta-Tat-6His

2019-08-24T15:51:28Z

Crm: Created page with "File:2580 2579 digestion.JPG Category:Cloning"

[[File:2580 2579 digestion.JPG]]
[[Category:Cloning]]

2595 pDB.His.GST-D7324

2019-08-23T23:53:27Z

Crm: Created page with "File:2593 digestion.JPG Category:Cloning"

[[File:2593 digestion.JPG]]
[[Category:Cloning]]

2586 pET28a-6H-D7324-AP205

2019-08-23T17:21:28Z

Crm: Created page with "File:2586 digeastion.JPG Category:Cloning"

[[File:2586 digeastion.JPG]]
[[Category:Cloning]]

2579 pET28a-6H-Tat-AP205

2019-08-23T16:43:57Z

Crm:

[[File:8 15 19 digestion1.JPG|RTENOTITLE]]

[[File:8 15 19 digestion2.JPG|RTENOTITLE]]

2579-3 is parental

8/22/19

[[File:2580 2579 digestion.JPG|RTENOTITLE]]

2579 #6 is good by sequencing
[[Category:Cloning]]

Commercial Enzymes

2019-08-21T22:41:53Z

Crm:

{| border="1" cellspacing="1" cellpadding="1" style="width: 970px;"
|-
| 
| 
| 
| 
| 
| 
| style="width: 99px;" | 
| style="width: 58px;" | 
| style="width: 58px;" | 
| 
| style="width: 285px;" | 
| style="width: 50px;" | 
|-
| Excellgen Cat #
| Clone #
| Size
| Price
| Name
| Supplier Cat #
| style="width: 99px;" | Supplier
| style="width: 58px;" | Lot 
| style="width: 58px;" | Concentration
| Size
| style="width: 285px;" | Storage Buffer
| style="width: 50px;" | Price
|-
| EG-150
| 
| 100units, 10U/ul, 10ul
| 
| 
| 
| style="width: 99px;" | 
| style="width: 58px;" | 
| style="width: 58px;" | 
| 
| style="width: 285px;" | 
| style="width: 50px;" | 
|-
| EG-151 
| 
| 2,000 U/ml,50ul, 100 units
| $85
| PolyU polymerase,   Schizosaccharomyces pombe 
| M0337S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 0.34 mg/ml
| 2000U/ml, 30 ul, 60 units
| style="width: 285px;" | 
| style="width: 50px;" | $85
|-
| EG-1057 
| 1942 
|
100,000 U/ml

100U/ul

1ug/ul, 100 ul, 100ug

| $350
| phi29 (φ29) DNA Polymerase 
| EP0094 
| style="width: 99px;" | Thermofisher
| style="width: 58px;" | 
| style="width: 58px;" | 0.1ug/ul, 10u/ul, 
|
5000U

500 ul -->

50 ug

50 ul of 1 mg/ml stock + 450 ul buffer

| 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20 and 50% (v/v) glycero
| $826
|-
| 
| 
| 
| 
| E coli polyA polymeras
| M0276S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 
| 20ul, 7.43 mg/ml by reading but has 0.1% Triton X-100
| 
| 
|-
| 
| 
| 
| 
| T4 DNA ligase , 400units/ul
| M0202L
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 
|
1.1mg/ml

0.76mg/ml (1271711)

| 
| 
|-
| 
| 
| 
| 
| T4 DNA ligase. 2U/ul
| F93301
| style="width: 99px;" | Lucigen
| style="width: 58px;" | 
| style="width: 58px;" | 
| 2.7mg/ml
| 
| 
|-
| 
| 
| 
| 
| T4 DNA ligase. 2U/ul 
| 46-0117
| style="width: 99px;" | invitrogen
| style="width: 58px;" | 
| style="width: 58px;" | 
| 0.61mg/ml
| 
| 
|-
| 
| 
| 
| 
| T4 DNA ligase. 5U/ul 
| K-1232
| style="width: 99px;" | Thermofisher
| style="width: 58px;" | 00680547
| style="width: 58px;" | 
| 1.03mg/ml
| 
| 
|-
| 
| 
| 
| 
| Tag Ligase, 40,000U/ml
| M0208S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 
| 0.3/mg/ml
|  50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 µg/ml BSA and 50% glycerol. 
| 
|-
| 
| 
| 
| 
| Bst polymerase, large fragment
| M0275S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 8000U/ml
|
2.32mg/ml

not accurate because Tx-100

|
storage buffer has Triton X-100

10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.1 @ 25°C

based on NEB's specific Activity: 120,000 units/mg,  100U/ul = 20,000U/200ul, 90% purity, then need 200 ug protein in 200 ul., take 10 ul of 29mg/ml-->200 ul storage buffer, protein conc is 1.7 mg/ml

| 
|-
| 
| 
| 
| 
| T3
| M0317S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 3000U/ul
| 2mg/ml
| 
| 
|-
| EG-160
| 
| 
| 
| T4 PDG
| M0308S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 10U/ul
| 3.93mg/ml
| storage buffer has 0.15% Triton X-100, do not know exact protein conc
| 
|-
| EG-1005
| 
| 
| 
| APE 1
| M0282S
| style="width: 99px;" | NEB
| style="width: 58px;" | 
| style="width: 58px;" | 10U/ul
| 1.24
| 
| 
|}
[[Category:Cloning]]

File:2586 2596 digestion.JPG

2019-08-21T20:30:50Z

Crm:

2570 pDB-His-MBP-Tth-Muts

2019-08-21T01:09:41Z

Crm: Created page with "File:2570 subcloning.JPG digestion of miniprep, 8/20/2019 File:2570 digestion.JPG looks like clone # 1,2,3 are good"

[[File:2570 subcloning.JPG]]

digestion of miniprep, 8/20/2019

[[File:2570 digestion.JPG]]

looks like clone # 1,2,3 are good

2582 pJet1.2-QbetaPac-TagGFP

2019-08-18T22:11:53Z

Crm:

[[File:8 15 19 digestion1.JPG|RTENOTITLE]]

http://www.excellgen.net/eln/images/0/0d/8_15_19_digestion2.JPG

[[File:2595 2582 digestion.JPG]]
[[Category:Cloning]]

File:2595 2582 digestion.JPG

2019-08-18T22:10:06Z

Crm:

File:2596 2567 digestion.JPG

2019-08-18T22:09:53Z

Crm:

2593 pDB.His.GST-SpyCatcher-AP205cp3

2019-08-13T13:47:02Z

Crm:

[[File:2593 2581 2582 digestion.JPG|RTENOTITLE]]

sequencing result, clone #4 is good, make grlycerol stock

File:2593 2581 2582 digestion.JPG

2019-08-12T17:55:36Z

Crm:

2555 pET28a-Qbeta-CETP-N13-Avitag-6His

2019-08-08T14:03:04Z

Crm: Created page with "File:2555 8 7 19.JPG"

[[File:2555 8 7 19.JPG]]

File:2555 8 7 19.JPG

2019-08-08T14:01:30Z

Crm: Crm uploaded a new version of File:2555 8 7 19.JPG

971 pET28a Spycatcher-AP205cp3

2019-08-08T13:57:34Z

Crm: Created page with "Protein Purification from Pellet (8M Urea soluble fraction) File:971 P 8 8 19.JPG Category:Cloning"

Protein Purification from Pellet (8M Urea soluble fraction)

[[File:971 P 8 8 19.JPG]]
[[Category:Cloning]]

File:971 P 8 8 19.JPG

2019-08-08T13:52:44Z

Crm:

2503, 2535, 971 2566, 2562 824 2558 protein purification

2019-08-07T14:24:10Z

Crm:

[[File:Jpg065.jpg|RTENOTITLE]]

1- 2503

2- 2535 

3- 971#7

4- 2566

5- 2562 #1

6- 2562 #1

7- 824 #6

8- 2558 #8

9- Page Ruler Plus
[[Category:QC]]

File:Jpg065.jpg

2019-08-07T14:12:01Z

Crm:

Purification of VLP

2019-07-20T16:13:33Z

Crm:

== Medium and Buffers. ==

'''LB'''

500 g LB powder, 80 g Na2HPO4.7H2O, 10 g KH2PO4, add 20 L ddH2O, aliquote  1 L medium into 2 L culture flask, autoclave

'''50X 5052'''

250 g glycerol, 25 g glucose, 100 g alpha lactose, add ddH2O to 800 ml, stir until dissolve, add ddH2O to 1 L, autoclave

'''High Salt Buffer A (the same as VLP Lysis Buffer)'''

20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM Imidazole, 0.1% NP-40 or 0.1% Triton X100

'''Low Salt Buffer B'''

20 mM KPO4, pH 7.4, 50 mM NaCl, 300 mM Imidazole

'''Buffer 2'''

20 mM Tris-HCl, pH 8.0, 50 mM NaCl.

'''Buffer B for XC'''

20 mM KPO4, pH7.0 or pH 7.4,  1.5 M NaCl

For VLP with pI >8, use pH 7.4, for pI <8, use pH 7.0

'''VLP Lysis Buffer'''

20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM Imidazole, 0.1% NP-40 or 0.1% Triton X100

'''Inclusion Body Solubilization Buffer'''

20 Tris-HCl, pH 8.0, 8 M Urea, 20 mM Imidazole, 50 mM NaCl

== Culture and Harvest Cells ==

1, Streak a VLP clone in Nico21(DE3)pLysN  onto a LB/KC plate, label the plate (write clone #), grow at 37 oC incubator O/N, agar side up

2, Wash E coli into an autoclaved glass culture tube with 15 ml LB/KC medium

3. Culture cells for 1 hour at 37 oC in a shaker incubator

4. Pour 15 ml the above starter stock into 1 L LB/KC medium, add 20 ml of 50X 5052 using autoclaved 100 ml glass graduated cylinder

5. Grow cells to OD600  0.6 ~ 1.0

6. Add 0.5 ml 1 M IPTG, induce at 37 oC for 4 to 6 hours. You can also leave the induction overnight '''providing you culture  the cells overnight at 16 oC in the cold room downstairs'''. Sometimes cells may be lysed during overnight culture. So it would be better to spin down cells after 4 hours induction.

7. Spin down cells, 5 min at 5000 rpm using a high speed centrifuge.

8. Combine cells into one centrifuge tube, use LB/KC medium (supernatant liquid from previous spin) to wash cells into one tube. Spin again to collect pellet.

9. Record wet cell weight. Label tube

10. Store cells (tube) at -80 oC freezer for 1 hour or overnight.

== Purification ==

11 .Warm up cell pellet, add 200 ml VLP Lysis Buffer.

12. Sonicate 2 min (do not use homogenizer as it is more efficient to use sonicator for VLP).

13. Spin using ultra centrifuge, 30,000 rpm, 4 oC, 30 min.

14. Combine supernatant into one 250 ml  tube. Label as (Clone#) S

15. Combine pellet into another tube using  150 ml  Inclusion Body Solubilization Buffer, label as (Clone#) P

16. Load supernatant (Clone#) S  into one 5 ml Ni FF column in fridge, label the Ni column as (Clone#) S 

17. Load (Clone#) P solution into another 5 ml Ni FF column at room temperature,  label the Ni column as (Clone#) P

18. Wash both Ni FF columns with 100 ml '''High Salt Buffer A'''.

19. Use appropriate connectors, connect Ni FF column to AKTA Prime Plus FPLC, connect 2 X 1 ml Q FF and 1 ml SP HP column to the outlet of  Solenoid valve

20. Test run using '''program 10''' on PC. Check if there is any leaks, and check computer if it is collecting data

21. Purify protein using AKTA Prime Plus,   program '''6''' on PC, elute protein directly into 2X 1 ml Q FF + 1 ml SP HP.

Buffers: '''High Salt Buffer A''', '''Buffer 2''', '''Low Salt Buffer B'''

22. Disconnect Ni FF column, <s>move 2 X 1ml Q FF + 1 ml SP HP to FPLC.</s>

23. Test run using program 10 on PC, Check if there is any leaks, and check computer if it is collecting data.

<s>24. Switch out the tube rack with a clean new one </s>

<s>24. Put the red columns where on the platform and transfer the label  from the green column to the red ones</s>

24. Check for leaks with program 10 and don't forget to switch the Buffer B to XC

25. Purify protein further using AKTA Prime Plus,   program '''9''' on PC. Collect fractions. 

Buffers: '''Buffer 2,''' '''Buffer B for XC'''

26. Measure protein concentration in peak tubes using nanodrop 

27. Take 30 ul solution + 10 ul 4X SDS-PAGE Loading Buffer, boil 10 min, and run SDS-PAGE gel to check purity.

28. Label and store tubes with protein in 4 oC fridge.
[[Category:Protocols]]

File:2558 7 19 19.png

2019-07-19T16:00:38Z

Crm:

[[Category:Protein Purification]]

File:71819.jpg

2019-07-18T16:43:39Z

Crm:

[[Category:Customers]]

Customer service projects for Syd Inc 0710-17-2019

Lane 1, Histagged Ag, 1.9 mg/ml, 11 ml (6 x 1 ml was shipped to Syd 071719, remained 5 ml was stored at -80C)

Lane 2 Histagged Ag, 2.2 mg/ml, 11 ml (stored at -80C)

The histagged protein was purified by Ni affinity on an 5ml Hitrap Ni column by stepwise imidazole gradient wash and elution. The i300 mM eluted fractions were pooled and dialyzed againise PBS containing 10% glycerol at 4C.

Lane 3, FBL, 1.3 mg/ml, total 5.5 ml (5 x 1 ml in 20% glycerol Hepes buffer, 1 mM EDTA, 500 mM NaCl, 5 mM DTT and 0.05% NP40) frozen at -80C before shipping to Syd via dryice shipment on 071719. (service fee charged $2,170)

File:Hitagged Ag and FBL 071719 prep.xcf

2019-07-18T16:30:25Z

Crm:

File:2565SP71419.png

2019-07-18T14:26:53Z

Crm:

2560, 2038, 2565, 2571

2019-07-17T15:42:14Z

Crm:

                                                                 5     6      7      8      9

[[File:71619.png|RTENOTITLE]]

{| class="wikitable"
|-
! Lane
! ID
! Name
! Host
! pI/MW
! Induction Condition
! Expressed?
|-
| 4
|
| kDa
| 6.5 14.4 21.5 31 45 66.2 97.4 116 200
|
|
|
|-
| 1
| 2560
| pET28a-CETP-N1-AP205
| Nico(DE3)pLysN
|
| 37 oC for 4 hours
| yes
|-
| 2
| 2038
| pL42-sso7d-KlenTaq
| Nico(DE3)pLysN
|
| 37 oC for 4 hours
| yes
|-
| 3
| 2565
| pET29b-MT-MMLV
| Nico(DE3)pLysN
| 9.08 / 79.6
| 37 oC for 4 hours
| yes
|-
| 5,6,7,8,9
| 2571
| pET28a-FBL
| Nico(DE3)pLysN
|
| 37 oC for 4 hours
| yes
|}
[[Category:QC]]

File:71619.png

2019-07-17T14:55:51Z

Crm:

Lab Rules

2019-07-16T14:27:51Z

Crm:

== Laboratory Rules ==

#Employee should come to work on time (9 am or early).
#All experiments must be properly and promptly documented. All experimental steps must be recorded and all results must be organized in comprehensible formats. All experiments procedure and results should be uploaded to ELN.
#Document all failed experiments in details, upload all data including "not working" or unsatisfitory result due to  careless mistakes or unexpected errors to ELN.
#Person who deliberately falsified data will be punished by employment termination and possibly legal actions.
#Smoking, eating, and drinking are expressly forbidden and NOT allowed in the laboratory.
#Wear any required personal protective equipment (lab coat, gloves, goggles, etc).
#Wear impermeable shoes that completely cover the feet (i.e. no sandals, no clogs, and no fabric shoes) in areas where chemicals, biological, and radiological materials are present. Only shoes that provide complete foot covering are allowed in the lab. Long hair should be pulled back. Shorts, skirts, dresses, and tight-fitting clothes are not allowed in the lab.
#Discussion of topics related to sex, age, race, religion, salary, personal or family matters are expressly forbidden and NOT allowed in the lab.
#Volatile, toxic, corrosive, and/or irritating chemicals must be handled in the fumehood.
#Avoid breathing, tasting, or having skin in contact with chemicals.
#Never pipet by mouth. Always use a suction bulb or pipet aid.
#No unauthorized experiments are to be performed in the laboratory. Never leave your experiment unattended unless authorized to do so.
#Work areas must be left clean and dry prior to leaving the lab. Enzymes, chemicals and reagents must be returned to their proper places.
#You should always wash your hands before leaving the laboratory. If you are the last person to leave the lab you must check the entire lab: turn off electrophoresis power supply, unplug nanodrop, turn off microplate reader etc, then lock the door.
#Do not eat foods containing onions for breakfast and lunch, drink enough water during the day
#Make cell phone vibrate before starting an experiment. Answering phone calls from friends and family during experiment is not allowed.
#Listening to iPod or other music devices with earphones is prohibited while doing experiment.
#You should deduct your time from timesheet if you spend more than 20 minutes on lunch.
#Do not wear gloves when using: computer, gel-documentation camera, microscope or open the lab doors.
#Do not use lab computer to write personal emails.
#Do not read news articles using laboratory computers.
#Do not use protocols downloaded from internet without authorization.
#Remove one glove when handling common equipment.
#Personal electronics such as IPad, iPhone, IPod, earphone, and computers are not allowed in the lab.
#Eat breakfast before coming to work.

Last Update: July 16, 2019
[[Category:Employees]]

Inclusion body purification

2019-07-12T18:55:55Z

Crm:

Grow bacteria overnight in -80 oC freezer

Homogenize with buffer A (optional)

Harvest  by centrifugation (optional)

Resuspend in 50 mM NaH2PO4, 150 mM NaCl, 10 mM MgCl2, 0.25% Triton X-100, pH 7.2

Digest nucleic acids   with 1500 U Benzonase (Sigma-Aldrich) for 1 h  at room temperature

Harvest inclusion bodies containing recombinant protein by centrifugation (optional)

Three washes with 100 mM Tris-Cl, 5 mM EDTA, 5 mM DTT, 2% Triton X-100, pH 7.0

Solubilize inclusion bodies in 8 M urea, 100 mM Tris-Cl, 100 mM DTT, pH 8.0

If not fully dissolved, use filter and/or biorad to filter out the supernatant

Load on a Ni-NTA column (Qiagen), which has been previously equilibrated with 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-Cl, 2 mM β-mercaptoethanol, pH 8.0.

Elute bound protein   with 8 M urea, 100 mM NaH2PO4, 10 mM Tris, 2 mM β-Mercaptoethanol pH 4.5

Dialyse against 2 M urea, 50 mM NaH2PO4, 0.5 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 10% glycerol, pH 8.5.

Refold protein  by stepwise dialysis against

50 mM NaH2PO4, 0.5 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 10% glycerol, pH 8.5,

then against 50 mM NaH2PO4, 10% glycerol, pH 8.5.
[[Category:Protocols]]

CETP VLP Expression Screening

2019-07-12T14:43:06Z

Crm:

expression Screening  in urea soluble fraction (inclusion body)

[[File:71119VLPscreening.jpg|RTENOTITLE]]

{| class="wikitable"
|-
! Lane
! ID
! Name
! Host
! pI/MW
! Induction Condition
! Expressed?
|-
| M
| Protein Marker
| PageRuler Plus
| 10 15 25red 35 55 70red 100 130 250
|
|
|
|-
| 1
| 2557
| pET28a-Qbeta-CETP-N1-Avitag-6His
| Nico(DE3)pLysN
| 7.86 / 19.6
| 37 oC for 4 hours
| yes, IB
|-
| 2
| 2558
| pET28a-Qbeta-CETP-N2-Avitag-6His
| Nico(DE3)pLysN
| 6.43 / 20.2
| 37 oC for 4 hours
| yes, IB
|-
| 3
| 2559
| pET28a-Qbeta-CETP-N3-Avitag-6His
| Nico(DE3)pLysN
| 7.13 / 20.2
| 37 oC for 4 hours
| yes, IB
|-
| 4
| 2555
| pET28a-Qbeta-CETP-N13-Avitag-6His
| Nico(DE3)pLysN
| 7.87 / 22.4
| 37 oC for 4 hours
| yes, IB
|-
| 5
| 2556
| pET28a-Qbeta-CETP-N12-Avitag-6His
| Nico(DE3)pLysN
| 6.75 / 22.6
| 37 oC for 4 hours
| yes, IB
|-
| 6
| 2560
| pET28a-CETP-N1-AP205
| Nico(DE3)pLysN
| 8.98 / 17.8
| 37 oC for 4 hours
| yes, IB
|-
| 7
| 2561
| pET28a-CETP-N2-AP205
| Nico(DE3)pLysN
| 7.06 / 18.3
| 37 oC for 4 hours
| yes, IB
|-
| 8
| 2562
| pET28a-CETP-N3-AP205
| Nico(DE3)pLysN
| 8.65 / 18.3
| 37 oC for 4 hours
| yes, IB
|-
| 9
| 2563
| pET28a-CETP-N12-AP205
| Nico(DE3)pLysN
| 7.85 / 20.6
| 37 oC for 4 hours
| yes, IB
|-
| 10
| 2564
| pET28a-CETP-N13-AP205
| Nico(DE3)pLysN
| 8.95 / 20.6
| 37 oC for 4 hours
| yes, IB
|-
| 11
| 971
| pET28a-SpyCatcher-AP205cp3
| Nico(DE3)pLysN
| 6.50 / 26
| 37 oC for 4 hours
| yes, IB
|-
| 12
| 2565
| pET29b-MT-MMLV
| Nico(DE3)pLysN
| 9.08 / 79.6
| 37 oC for 4 hours
| yes, more  as inclusion body
|-
| 13
| 2566
| pET29a-Qbeta-SpyCatcher-Avitag-6His
| Nico(DE3)pLysN
| 6.20 / 27.7
| 37 oC for 4 hours
| yes, IB
|-
| 14
| 2567
| pET29b-RT-Pol
| Nico(DE3)pLysN
| 6.80 / 101.5
| 37 oC for 4 hours
| yes, not much
|-
|
|}
[[Category:QC]][[Category:Protein Expression]]

File:71119VLPscreening.jpg

2019-07-12T13:39:24Z

Crm:

File:7 11 2019 VLP Purification.jpg

2019-07-11T18:26:37Z

Crm:

Taq Buffer

2019-07-08T13:47:44Z

Crm:

== NEB Taq Buffer, 10 X ==

100 mM Tris-HCl, 500 mM KCl, and 15 mM MgCl2, pH 8.3 @ 25°C.

'''1'''. 100 ml of 1M Tris-HCl, pH 8.30 '''Trizma HCl: '''6.14 g '''Trizma Base: '''7.40 g

ultrapure water to 100 ml

'''2'''. 50 ml of 150 mM MgCl2 · 6H2O: MW 203.3

 1.53 g, add ultrapure water to 50 ml

'''3'''. 1% Tween 20:  5 g in 50 ml ultrapure water.

'''To prepare 50 ml 10 X Buffer without Tween 20:'''

5 ml 1M Tris-HCl, pH 8.30.

5 ml 150 mM MgCl2

0.05 * 0.5 * 74.55=1.86 g KCl.

Ultrapure Water to 50 ml.

'''To prepare 50 ml 10 X Buffer without Tween 20:'''

5 ml 1M Tris-HCl, pH 8.30.

5 ml 150 mM MgCl2

+ 0.05 * 0.5 * 74.55=1.86 g KCl.

+  0.5 ml of 10% Tween 20

ultrapure Water to 50 ml.

'''1 M Trehalose MW 342.29'''

17 g in 50 ml ultrapure water.

== 10X Taq Buffer with KCl ==

100 mM Tris-HCl (pH 8.8 or 8.3 at 25°C), 500 mM KCl, 15 mM MgCl2, 0.8% (v/v) Nonidet P40 or Triton X-100.

== 10X Taq Buffer with (NH4)2SO4 ==

750 mM Tris-HCl (pH 8.8 at 25°C), 15 mM MgCl2, 200 mM (NH4)2SO4, 0.1% (v/v) Tween 20.

2X Taq MasterMix

For StepOne ABI

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| Components 
| ul 
|-
| H2O 
| 15.15 
|-
| Buffer 10 X 
| 2.5 
|-
| MgCl2, 50 mM 
| 1.5 
|-
| dNTPs 10 mM 
| 0.5 
|-
| Primers 5 uM 
| 2 
|-
| BSA 10 mg/ml 
| 0.25 
|-
| Taq  5 U/ul 
| 0.2 
|-
| Sybr (10x) 
| 0.4 
|-
| Rox (5x) 
| 0.5 
|-
| cDNA 
| 2 
|}

Increase SYBR and decrease ROX concentrations for Stratagene Mx3005P and BioRad iCycler

Sigma Jump Start Taq Ready Mix. 5ul SYBR green mix MgCl2 1.2 ul 0.4 ul forward primer (10 pmol) 0.4 ul reverse primer (10 pmol) 1 ul water 2ul cDNA (1/4 diluted cDNA)

For High Resolution Melting: use Biotium EvaGreen  and Promega BRYT Green, becuase SYBR Green  inhibits PCR at higher concentrations. EvaGreen at high enough concentrations to saturate the double strand DNA

== Sigma AccuTaq LA and REDAccuTaq LA Buffer ==

AccuTaq LA: 500 mM Tris-HCl, adjust pH to 9.3 using NH4OH, 150 mM (NH4)2SO4, 25 mM MgCl2, 1% Tween 20

REDAccuTaq LA : 500 mM Tris-HCl, adjust pH to 9.3 using KOH, 150 mM (NH4)2SO4, 25 mM MgCl2, 1% Tween 20
[[Category:Buffer Recipe]]

2566 2555 2557 2567

2019-07-05T20:50:01Z

Crm:

Date: 7/5/2019

2566, 2555, 2557, 2567

Marker: PageRkueer Plus

[[File:7 5 2019.jpg|RTENOTITLE]]
[[Category:QC]]

File:7 5 2019.jpg

2019-07-05T20:47:47Z

Crm:

Transfomation for protein expression

2019-07-03T14:05:37Z

Crm:

= Creating Transfomation for protein expression =
<div id="bodyContent" class="mw-body-content"><div id="contentSub"></div></div>
*take competent cells (Nico21(DE3)pLysN) from minus 80 freezer
*pipet 100 microliters and mix with 0.5 microliter of given plasmid
*incubate on ice for 30 minutes
*pour some water on the PCT PCR machine and heat up to setting "42-30" and put on it for about 30~45  seconds
*take it out and put on ice, let sit on ice for 2 minutes
*add 750 microliters of SOC
*put in shaker for about two hours
*spin for one minute
*dump out liquid but leave about 50-100 microliters
*suspend bacteria with pipette
*spray on LB/KC plate and spread with glass beads
*put in incubator overnight, agar side up
*---------> next day
*wash out colonies with LB/KC or LB/AC and pipette into a 50 mL plastic tube
*add LB/KC or LB/AC until it reaches 15 mL
*put in shaker for about 2-3 hours
*add to 1 or 2 Liters of LB/KC or LB/AC
*grow in shaker
*induce with 0.5 mL of 1 M IPTG per bottle
*induce at 37 oC for 4 hours, or room temperature overnight
*spin down cells, combine, weigh cell pellet weight and store cells at -80 freezer
[[Category:Protocols]]

Transfomation for making glycerol stock

2019-07-03T14:00:26Z

Crm:

== Creating Transfomation for making glycerol stock ==

*take competent cells from minus 80 freezer
*pipet 100 microliters and mix with 0.5 microliter of given plasmid
*incubate on ice for 30 minutes
*pour some water on the PCT PCR machine and heat up to setting "42-30" and put on it for about 30~45 seconds
*take it out and let sit on ice for 2 minutes
*add 750 microliters of SOC
*put in 37 oC shaker incubator for about two hours
*spin for 30 seconds
*dump out liquid but leave about 50-100 microliters
*then spray on LB/K plate and incubate
*---------> next day
*wash out colonies with 1.5 ml   <s></s>LB/K
*spin down, remove ~0.75 ml liquid
*add .75 mL of 80% glycerol
*print label and enter into database
*put into a freezer box in -80 freezer
[[Category:Protocols]]

2569 pFastbac1-Flpo

2019-06-30T02:41:19Z

Crm: Created page with "File:2569 digestion.JPG Category:Cloning"

[[File:2569 digestion.JPG]]
[[Category:Cloning]]

File:2569 digestion.JPG

2019-06-30T02:40:17Z

Crm:

File:Cre 3ml heparin 5 30 19.JPG

2019-05-30T20:35:30Z

Crm:

File:T4PNK SP.JPG

2019-05-25T23:37:34Z

Crm: