ELN - User contributions [en]

546-pBad24-sfGFP

2012-02-17T19:53:16Z

Ruili.su:

== '''Cloning of 546-pBad24-sfGFP''' ==

'''Purpose:''' Cloning of 546-pBad24-sfGFP by genesynthesis

'''Date: '''2012-2-7--

'''Notebook:003732'''

'''Page:'''

'''Clone ID:546'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-2-7

[[Image:546+1004 PCR 2-8-2012.JPG]] 

 

PCR template is Ecoli-sfGFP1 to Ecoli-sfGFP20 

Primer-F Ecoli-sfGFP1 

Primer-R Ecoli-sfGFP20

[[Image:546+1002+1005 clone 2-15-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 305px; height: 96px;"
|-
| 
| band 3 
| band 4 
|-
| 
| PCR product 
| pBad24 545 
|-
| digest enzymes 
| colspan="2" |         EcoR1 + HindIII 
|}

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 7px; height: 23px;"
|-
| 
|}

=== Colony Screening ===

Date: 2012-2-17

Make miniprep and digest with EcoR1 + HindIII 

[[Image:546+1005_digest_detect_2-17-2012.JPG]]

clone 1,2,3,5,6 may be right ,need to sequence

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

{| cellspacing="1" cellpadding="1"
|-
| 
|}

 

{| cellspacing="1" cellpadding="1"
|-
| 
|}

 

{| cellspacing="1"
|-
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|}

[[Category:Cloning]]

File:546+1005 digest detect 2-17-2012.JPG

2012-02-17T19:49:06Z

Ruili.su:

File:1002 digest detect 2-17-2012.JPG

2012-02-17T19:48:54Z

Ruili.su:

File:1009 clone 2-16-2012.JPG

2012-02-16T18:39:39Z

Ruili.su:

File:1002 PCR 2-14-2012.JPG

2012-02-15T20:55:00Z

Ruili.su:

File:546+1002+1005 clone 2-15-2012.JPG

2012-02-15T20:28:43Z

Ruili.su:

File:1008+1009 digest detect 2-15-2012.JPG

2012-02-15T20:24:42Z

Ruili.su:

File:1008+1009 clone 2012-2-13.JPG

2012-02-13T19:48:59Z

Ruili.su:

File:1008+1009 PCR 2012-2-10.JPG

2012-02-13T19:48:46Z

Ruili.su:

File:534 clone 2012-2-6.JPG

2012-02-10T19:57:07Z

Ruili.su:

323-pLenti-EF1-MCS

2012-02-10T19:56:17Z

Ruili.su:

== '''Cloning of 323-pLenti-EF1-MCS''' ==

'''Purpose:''' Cloning of 323-pLenti-EF1-MCS

'''Date: '''2011-12-28

'''Notebook:'''

'''Page:'''

'''Clone ID:323'''

'''Experimenters:''' yingying

== '''Results''' ==

'''[[Image:323 PCR 12-29-2011.JPG]]''' 

'''template: pPuroGreen 178''' 

Primer-F EF1-<strike>NdeI</strike>-F agct GCTAGC c gtgaggctcc ggtgcccg Primer-R EF1-BamH1-R (cttccatttcaggtgtcgtga GGATCC atcg) cgatGGATCCtcacgacacctgaaatggaag 

[[Image:169+323+459 clone 12-29-2011.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 393px; height: 96px;"
|-
| 
| band 3 
| band 4 
|-
| 
| PCR product 
| pLenti-Plus (152) 
|-
| digest enzyme 
| colspan="2" |          <strike>Nde1</strike> + BamH1 
|}

 

=== Subcloning ===

Date: 

 

=== Colony Screening ===

Date: 2012-1-3 

Make miniprep and digest with <strike>Nde1</strike> + BamH1 

[[Image:169+323 digest detect 1-3-2012.JPG]]

  '''Result intepretation/Summary'''

== ==

== '''Conclusion''' ==

=== ===

 --[[User:Yingying.lu|Yingying.lu]] 15:05, 29 December 2011 (EST)

 

== '''Cloning of 323-pLenti-EF1-MCS''' ==

'''Purpose:''' Cloning of 323-pLenti-EF1-MCS

'''Date: '''2012-1-4

'''Notebook:'''

'''Page:'''

'''Clone ID:323'''

'''Experimenters:''' yingying

== '''Results''' ==

[[Image:323 PCR 1-4-2012.JPG]] 

'''template: pPuroGreen 178''' 

Primer-F EF1-<strike>NdeI</strike>-F agct GCTAGC c gtgaggctcc ggtgcccg Primer-R EF1-BamH1-R (cttccatttcaggtgtcgtga GGATCC atcg) cgatGGATCCtcacgacacctgaaatggaag 

[[Image:323 clone 1-4-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 393px; height: 96px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| pLenti-Plus (152) 
|-
| digest enzyme 
| colspan="2" |          <strike>Nde1</strike> + BamH1 
|}

 

=== Subcloning ===

Date: 

 

=== Colony Screening ===

Date: 2012-1-6 

Make miniprep and digest with <strike>Nde1</strike> + BamH1 

[[Image:323 digest detct 1-6-2012.JPG]]

 

  '''Result intepretation/Summary'''

== ==

== '''Conclusion''' ==

=== ===

 --[[User:Yingying.lu|Yingying.lu]] 15:05, 29 December 2011 (EST)

== '''Cloning of 323-pLenti-EF1-MCS''' ==

'''Purpose:''' Cloning of 323-pLenti-EF1-MCS by PCR 

'''Date: '''2012-1-26--

'''Notebook:'''

'''Page:'''

'''Clone ID:323'''

'''Experimenters:''' yingying

== '''Results''' ==

 

=== cloning ===

Date: 2012-1-26

[[Image:544+323 PCR 1-26-2012.JPG]] 

Primer-F EF1-NdeI-F agct CATATG c gtgaggctcc ggtgcccg Primer-R EF1-BamH1-R (cttccatttcaggtgtcgtga GGATCC atcg) cgatGGATCCtcacgacacctgaaatggaag 

[[Image:323+534 clone 1-27-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 426px; height: 80px;"
|-
| 
| band 3 
| band 4 
|-
| 
| PCR product 
| 152 pLenti-plus 
|-
| digestion enzymes 
| colspan="2" |             Nde1 + BamH1 
|}

 

=== Colony Screening ===

Date: 2012-1-30 

Make miniprep and digest with  Nde1 + BamH1 

[[Image:323+534+535 digest detect 1-30-2012.JPG]] 

clone 1,2,5 may be right, need to sequence

 

 

 

== '''Result intepretation/Summary''' ==

*sequencing :no priming ,need to clone again 

== '''Conclusion''' ==

 

=== ===

 

== '''Cloning of 323-pLenti-EF1-MCS''' ==

'''Purpose:''' Cloning of 323-pLenti-EF1-MCS by PCR 

'''Date: '''2012-2-7--2012-2-10

'''Notebook:003732'''

'''Page:'''

'''Clone ID:323'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-2-7

[[Image:323 PCR 2-7-2012.JPG]] 

template:178- pGreenPuro(EF1) 

 

Primer-F EF1-NdeI-F agct CATATG c gtgaggctcc ggtgcccg Primer-R EF1-BamH1-R (cttccatttcaggtgtcgtga GGATCC atcg) cgatGGATCCtcacgacacctgaaatggaag

[[Image:323+1004 clone 2-8-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 384px; height: 96px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| 152 pLenti-plus 
|-
| digest enzymes 
| colspan="2" | Nde1 + BamH1 
|}

 

 

{| cellspacing="1" cellpadding="1" border="1"
|-
| 
|}

=== Colony Screening ===

Date: 2012-2-10

Make miniprep and digest with Nde1 + BamH1 

[[Image:323+1004_digest_detect_2-10-2012.JPG]]

 no insert 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

{| cellspacing="1" cellpadding="1"
|-
| 
|}

 

{| cellspacing="1" cellpadding="1"
|-
| 
|}

 

{| cellspacing="1"
|-
| 
|}

[[Category:Cloning]]

File:323+1004 digest detect 2-10-2012.JPG

2012-02-10T19:54:03Z

Ruili.su: uploaded a new version of "File:323+1004 digest detect 2-10-2012.JPG"

File:534+548 digest detect 2-9-2012.JPG

2012-02-10T19:53:45Z

Ruili.su: uploaded a new version of "File:534+548 digest detect 2-9-2012.JPG"

File:1005 PCR 2-10-2012.JPG

2012-02-10T17:29:48Z

Ruili.su:

548-PLVX-plus2-DIO-sfGFP

2012-02-10T17:23:48Z

Ruili.su: moved 548-PLVX-plus2-DIO-sfGFP to 1007-pLVX-loxP-sfGFP

#REDIRECT [[1007-pLVX-loxP-sfGFP]]

File:PKAalpha--02-07-2012.jpg

2012-02-10T15:19:41Z

Ruili.su:

File:323+1004 clone 2-8-2012.JPG

2012-02-08T17:10:17Z

Ruili.su:

File:546+1004 PCR 2-8-2012.JPG

2012-02-08T15:30:07Z

Ruili.su:

File:323 PCR 2-7-2012.JPG

2012-02-08T15:29:55Z

Ruili.su:

File:548 clone 2-7-2012.JPG

2012-02-08T15:29:41Z

Ruili.su:

Cre pTriEx-HTNC-purification

2012-02-07T20:52:33Z

Ruili.su:

Date: 2011-11-14 to 2011-11-17

Notebook: Ruili Su-- Protein purification

Page:002857 to 002859

Title: pTriEx-HTNC (Clone ID298) protein expression and purification.

[[Image:Cre (pTriEx-HTNC)-11-16-2011.jpg|107x294px|Cre (pTriEx-HTNC)-11-16-2011.jpg]]

 

PTriEx-HTNC: use plasmid (Clone ID298) to transform with Rosetta BL21 (DE3) pLysS competent cells and culture it on plate containing Chl/Car. Grow 1L at 30oC for O/N, then induce for 6 ~7 hours by IPTG at 30oC. The bacteria growth takes one night and one day. 

The sample is just from the washed procedure with washing buffer (50mM imidazole) and still not very pure. So, the imidazole concentration needs to be adjusted before the protein is completely eluted.

----

----

 

[[Image:PTriEx-HTNC(Cre) protein----Jan.03 2012.jpg|303x260px|PTriEx-HTNC(Cre) protein----Jan.03 2012.jpg]]

Notebook: ruili su 2

Page: 003487

Date: 01/03/2012

PTriEx-HTNC transformed with C41 (DE3)PlysS. After FPLC, runing the SDS-PAGE.

----

'''Result'''

The expression level is good and the purity of Cre protien is good as well.

[[Category:Protein_Purification]]

Taq Purification-pET30b-sso7d-Taq d289-con.

2012-02-07T20:48:20Z

Ruili.su:

[[Image:Taq_377_PCR_for_activity_02-04-_2012.JPG|136x218px]] 

Notebook: Ruili Su2

Page:003507

Date: 02/03--02/04/2012

After aliquote Taq and storing the product at -20 and -80 degree, runing PCR to check the activity of PCR.

----

'''Result'''

The low temprature as -20 and -80 are available to store Taq Polymerase, so the store condition is credible.

 

[[Category:Protein_Purification]]

File:Taq 377 PCR for activity 02-04- 2012.JPG

2012-02-07T20:39:42Z

Ruili.su:

PKA alpha pSpeedET-purification

2012-02-07T20:19:45Z

Ruili.su:

[[Image:PKA alpha-PspeedET-02-04-2012.jpg|275x274px|PKA alpha-PspeedET-02-04-2012.jpg]]

Notebook: Ruili Su 2 

Page:003504--003505 

Date:01/27/2012--02/01/2012 

PKAalpha-pSpeedET transformed with C43(DE3)PlySs and after induced with 1ml 20% Arabinose/L and growing 3-4hours at 

35 0C. Run FPLC. 

After FPLC, loading 15ul sample/well for SDS-PAGE. 

Line1: Flow through 

Line2--- eluted PKA. 

 

[[Image:PKA alpha-PspeedET-02-05-2012.jpg|275x273px|PKA alpha-PspeedET-02-05-2012.jpg]]

Date: 02/02/2012--02/04/2012 

Page: 003506--003507 

PKAalpha-pSpeedET transformed with C41(DE3)PlySs. Culturing cocndition is same as PKAalpha-pSpeedET transformed with C43(DE3)PlySs. 

Run SDS-PAGE to check size and purity.

Line 1: Flow through (15ul)

Line 2----: Eluted PKA after FPLC (15ul/well).

----

'''Result''' 

PKAalpha-sPeedET has protein expression after transformed with C41(DE3)PlySs and C43(DE3)PlySs, but the expression level is not good.

 

[[Category:Protein_Purification]]

532-PET28b-mIL10

2012-02-06T18:29:08Z

Ruili.su:

== '''Cloning of PET28b-mIL10''' ==

'''Purpose:''' Subcloning of PET28b-mIL10 from 529 pLVX-mIL10-IRES-EGFP to PET28b 

'''Date: '''2012-2-1

'''Notebook:'''

'''Page:'''

'''Clone ID:532'''

'''Experimenters:''' yingying

== '''Results''' ==

== '''Subcloning''' ==

Date: 2012-2-1

[[Image:529 digest detect 1-31-2012.JPG]]

529-3 digested band:BamH1 + Xho1  as 532 insert 

[[Image:532 clone 2-2-2012.JPG]] 

band 2: digested vector: BamH1 + Xho1 

 

 

=== Colony Screening ===

Date: 2012-2-1 

Make miniprep and digest with  BamH1 + Xho1 

[[Image:532_digest_detect_2-1-2012.JPG]]

clone 1,5 may be right

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

=== ===

 

 

 

 

  

=== ===

 --[[User:Yingying.lu|Yingying.lu]] 

[[Category:Cloning]]

File:532 digest detect 2-1-2012.JPG

2012-02-06T18:27:38Z

Ruili.su: uploaded a new version of "File:532 digest detect 2-1-2012.JPG"

File:PKA alpha-PspeedET-02-05-2012.jpg

2012-02-06T15:57:49Z

Ruili.su:

File:PKA alpha-PspeedET-02-04-2012.jpg

2012-02-06T15:57:20Z

Ruili.su:

File:532 clone 2-2-2012.JPG

2012-02-02T20:25:52Z

Ruili.su:

File:Taq 377 & PKA 01-14-2012.jpg

2012-02-02T20:03:37Z

Ruili.su:

529-pLVX-mIL10-IRES-EGFP

2012-01-31T19:30:05Z

Ruili.su: Created page with "'''Purpose:''' Subcloning of 529-pLVX-mIL10-IRES-EGFP by PCR '''Date: '''2012-1-27---- '''Notebook:''' '''Page:''' '''Clone ID:529''' '''Experimenters:''' yingying ..."

'''Purpose:''' Subcloning of 529-pLVX-mIL10-IRES-EGFP by PCR 

'''Date: '''2012-1-27----

'''Notebook:'''

'''Page:'''

'''Clone ID:529'''

'''Experimenters:''' yingying

== '''Results''' ==

=== Cloning ===

Date: 2012-1-28

[[Image:529+532_PCR_1-27-2012.JPG]] 

PCR using mIL10-BamH1-F + mIL10-Xho1-R, template is pBluescript SK-mIL10, 530 

[[Image:529+532_clone_1-27-2012.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 414px; height: 80px;"
|-
| 
| band 1 
| band2 
| 
|-
| 
| PCR product 
| pLVX-IRES-EGFP, 528 
| 
|-
| digest 
| colspan="3" |
                  BamH1 + Xho1 

|}

 

 

=== Colony Screening ===

Date:2012-1-31 

Make miniprep and digest with BamH1 + Xho1 

[[Image:529_digest_detect_1-31-2012.JPG]] 

clone 3 may be right, need to sequencing

 

  

== '''Result intepretation/Summary''' ==

== '''Conclusion''' ==

 

=== ===

 --[[User:Yingying.lu|Yingying.lu]] 

[[Category:Cloning]]

File:529 digest detect 1-31-2012.JPG

2012-01-31T19:23:08Z

Ruili.su:

File:529+532 clone 1-27-2012.JPG

2012-01-31T19:22:55Z

Ruili.su:

File:529+532 PCR 1-27-2012.JPG

2012-01-31T19:22:37Z

Ruili.su:

544-pTracer-SPbacCD

2012-01-30T19:31:34Z

Ruili.su: Created page with "== '''Cloning of 544-pTracer-SPbacCD''' == '''Purpose:''' Cloning of 544-pTracer-SPbacCD by PCR '''Date: '''2012-1-26-- '''Notebook:003719''' '''Page:''' '''Clone ID:54..."

== '''Cloning of 544-pTracer-SPbacCD''' ==

'''Purpose:''' Cloning of 544-pTracer-SPbacCD by PCR

'''Date: '''2012-1-26--

'''Notebook:003719'''

'''Page:'''

'''Clone ID:544'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-1-26

[[Image:542,543,544 PCR 1-26-2012.JPG]]

PCR template is SPbacCD 541

Primer-F SPbacCD-F AGGGAGACCCAAGCTGGCTAGC gcc acc ATG GAC GCC ATG CTG CGA GG Primer-R SPbacCD-R (GCTATTGACTACAAAAGGTGA GCGGCCGC TCGAGTCTAGAGGGCCCGT) ACGGGCCCTCTAGACTCGAGCGGCCGCTCACCTTTTGTAGTCAATAGC

[[Image:542,543,544 insert 1-27-2012.JPG]]

{| cellspacing="1" cellpadding="1" border="1" style="width: 288px; height: 80px;"
|-
| 
| band 3 
|-
| 
| PCR product 
|-
| digest enzymes 
| Nhe1 + Not1 
|}

 

[[Image:542,543,544 vector 1-27-2012.JPG]]

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 96px;"
|-
| 
| band 
|-
| 
| pTracer.CMV/Bsd 538 
|-
| digest enzymes 
| Nhe1 + Not1 
|}

 

{| cellspacing="1" cellpadding="1" border="1"
|}

=== Colony Screening ===

Date: 2012-1-30

Make miniprep and digest with Nhe1 + Not1

[[Image:542+543+544 digest detect 1-30-2012.JPG]]

clone 1,2,3,4,5 may be right, need to sequence

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==
 
{| cellspacing="1" cellpadding="1" border="1"
|}

{| cellspacing="1" cellpadding="1"
|-
| 
|}

[[Category:Cloning]]

543-pTracer-Pani2A

2012-01-30T19:26:40Z

Ruili.su: Created page with "== '''Cloning of 543-pTracer-Pani2A''' == '''Purpose:''' Cloning of 543-pTracer-Pani2A by PCR '''Date: '''2012-1-26-- '''Notebook:003719''' '''Page:''' '''Clone ID:543''..."

== '''Cloning of 543-pTracer-Pani2A''' ==

'''Purpose:''' Cloning of 543-pTracer-Pani2A by PCR

'''Date: '''2012-1-26--

'''Notebook:003719'''

'''Page:'''

'''Clone ID:543'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-1-26

[[Image:542,543,544 PCR 1-26-2012.JPG]]

PCR using Pani2A 540 as template

Primer-F Pani2A-F AGGGAGACCCAAGCTGGCTAGC gcc acc ATGGACGCCA TGCTGCGAGG Primer-R Pani2A-R (CAACCGAGGCGAGTGCTGA GCGGCCGC TCGAGTCTAGAGGGCCCGT) ACGGGCCCTCTAGACTCGAGCGGCCGCTCAGCACTCGCCTCGGTTG 

[[Image:542,543,544 insert 1-27-2012.JPG]]

{| cellspacing="1" cellpadding="1" border="1" style="width: 316px; height: 80px;"
|-
| 
| band 2 
|-
| 
| PCR product 
|-
| digest enzymes 
| Nhe1 + Not 1 
|}

 

[[Image:542,543,544 vector 1-27-2012.JPG]]

{| cellspacing="1" cellpadding="1" border="1" style="width: 308px; height: 96px;"
|-
| 
| band 
|-
| 
| pTracer.CMV/Bsd 538 
|-
| digest enzymes 
| Nhe1 + Not 1 
|}

 

{| cellspacing="1" cellpadding="1" border="1"
|}

=== Colony Screening ===

Date: 2012-1-30

Make miniprep and digest with Nhe1 + Not1

[[Image:542+543+544 digest detect 1-30-2012.JPG]]

clone 1,2,3,4,5 may be right, need to sequence

 

== '''Result intepretation/Summary''' ==

*

== '''Conclusion''' ==

 

=== ===

 

{| cellspacing="1" cellpadding="1" border="1"
|}

{| cellspacing="1" cellpadding="1"
|-
| 
|}

[[Category:Cloning]]

542-pTracer- BcCD

2012-01-30T19:21:22Z

Ruili.su:

== '''Cloning of 542-pTracer- BcCD''' ==

'''Purpose:''' Cloning of 542-pTracer- BcCD by PCR 

'''Date: '''2012-1-26--

'''Notebook:003719'''

'''Page:'''

'''Clone ID:542'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-1-26

[[Image:542,543,544_PCR_1-26-2012.JPG]] 

PCR using BcCD 539 as template 

Primer-F pBcCD-F, AGGGAGACCCAAGCTGGCTAGC gcc acc ATG TCT AAT AAT GCA CTC C Primer-R pBcCD-R, ACGGGCCCTCTAGACTCGAGCGGCCGCTCACCTTTTATAATCAATTGC 

[[Image:542,543,544_insert_1-27-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 311px; height: 80px;"
|-
| 
| band 1 
| 
|-
| 
| PCR insert 
| 
|-
| digest enzymes 
| Nhe1 + Not1 
| 
|}

[[Image:542,543,544_vector_1-27-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 290px; height: 96px;"
|-
| 
| band 
|-
| 
| pTracer.CMV/Bsd 538 
|-
| digest enzymes 
| Nhe1 + Not1 
|}

 

=== Colony Screening ===

Date: 2012-1-30

Make miniprep and digest with Nhe1 + Not1 

[[Image:542+543+544_digest_detect_1-30-2012.JPG]]

clone 1,2,3,4,5 may be right, need to sequence

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

=== ===

 

{| cellspacing="1" cellpadding="1" border="1"
|-
| 
|}

[[Category:Cloning]]

File:542+543+544 digest detect 1-30-2012.JPG

2012-01-30T19:16:05Z

Ruili.su:

File:542,543,544 vector 1-27-2012.JPG

2012-01-30T19:15:51Z

Ruili.su:

File:542,543,544 insert 1-27-2012.JPG

2012-01-30T19:15:38Z

Ruili.su:

File:542,543,544 PCR 1-26-2012.JPG

2012-01-30T19:15:20Z

Ruili.su:

File:323+534 clone 1-27-2012.JPG

2012-01-30T19:06:28Z

Ruili.su:

File:544+323 PCR 1-26-2012.JPG

2012-01-30T19:06:08Z

Ruili.su:

535-pAAV-CMV-DIO-sfGFP

2012-01-30T19:02:40Z

Ruili.su:

== '''Cloning of 535-pAAV-CMV-DIO-sfGFP''' ==

'''Purpose:''' Cloning of 535-pAAV-CMV-DIO-sfGFP by genesythesis 

'''Date: '''2012-1-24--

'''Notebook:'''

'''Page:'''

'''Clone ID:535'''

'''Experimenters:''' yingying

== '''Results''' ==

 

=== Cloning ===

Date: 2012-1-24

[[Image:535_PCR_1-24-2012.JPG]] 

gene synthesis, 

PCR1:DIO-sfGFP1 to DIO-sfGFP12 

PCR2:DIO-sfGFP13 to DIO-sfGFP26 

PCR: PCR1+PCR2 as template, 

primer:sfGFP1+DIO-sfGFP26 

[[Image:535_clone_1-25-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 422px; height: 83px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| 254 pAAV.CMV-MCS 
|-
| digestion enzymes 
| colspan="2" |         EcoR1 + BamH1 
|}

 

 

=== Colony Screening ===

Date: 2012-1-30 

Make miniprep and digest with  EcoR1 + BamH1 

[[Image:323+534+535_digest_detect_1-30-2012.JPG]] 

535-1,2,3,4,5,6 may right ,need to sequence

 

 

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

=== ===

 

[[Category:Cloning]]

File:323+534+535 digest detect 1-30-2012.JPG

2012-01-30T18:56:40Z

Ruili.su:

File:535 clone 1-25-2012.JPG

2012-01-30T18:56:26Z

Ruili.su:

File:535 PCR 1-24-2012.JPG

2012-01-30T18:56:10Z

Ruili.su:

Taq Purification-pET30b-sso7d-Taq d289

2012-01-26T23:12:56Z

Ruili.su:

[[Image:Protein 377 and Taq-MutS.jpg|259x214px|Protein 377 and Taq-MutS.jpg]] 

Date: 12/01/11---12/06/11 

Notebook: Ruili Su 002864----002868 

*377 protein: pET30b-sso7d-Taq-d289; the size between 70 KD and 55 KD; 

*Taq-Muts size is 128KD or so.

Line1: Taq-Muts flow through from Ni column; (10ul) 

Line 2: Taq-Muts washed with buffer A (10 mM Imidazole); (10ul) Line 3---6:Taq-Muts eluted with buffer B (300 mM Imidazole); (10ul) 

Line 7---10: 377 protein eluted with washing buffer (50 mM Imidazole); (10ul)  

Line 11: Taq enzyme from other. (5ul) 

'''Result'''

377 protein has correct size (line 7---10); also 50 mM Imidazole can comepletely elute the protein.

Taq-Muts has high level expression, it needs to pass through the Amylose beads to remove other bands.

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[[Image:Taq 377-12-14-2011.jpg|281x217px|Taq 377-12-14-2011.jpg]]

Date: 12/13/2011--12/14/2011 

Page: 002870-71 

Taq enzyme (377 protein) kept purifing by HiLoadTM 16/60 SuperdexTM 200

After this, concerntrate Taq and dilyze with buffer.

Lastly, need to measure the activity by PCR.  

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[[Image:Taq 377 PCR for activity 12-20-1022.JPG|215x284px|Taq 377 PCR for activity 12-20-1022.JPG]] 

Date: 12-22-2011 

Page: 002875 

run PCR to check the activity of Taq. 

1. 2mg/ml, store at 4 oC (1ul); 

2. 2mg/ml, store at -80 oC (1ul);

3. 4mg/ml, store at -80 oC (1ul); 

4. 2U/ul, control from product of Biodev, store at -20 oC (0.5ul). 

'''Result ''' 

Line 1 and line 3 could happen ?

By comparing line2 with line 4, 2mg/ml Taq should have 2u/ul activity. 

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[[Image:Taq 377 PCR for activity 1-12-2012.JPG|166x254px|Taq 377 PCR for activity 1-12-2012.JPG]] 

Date: 01-07-2012 through 01-12-2011 

Note Book: Ruili Su 2 

Page:003490-003492 

Run PCR to check Taq activity. 

Line1 : Taq washed from I20 ; 

Line2: Taq washed from I300 

Line3: Taq washed from I50 ; 

Line4: Taq from supertanant. 

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[[Image:Taq 377-01-17-2012.jpg|162x267px|Taq 377-01-17-2012.jpg]]

Date: 01-07-2012 through 01-13-2011

Note Book: Ruili Su 2

Page:003490-003493 

Run SDS-PAGE to check the purity of Taq.

Line1: supertanant;

Line2: Taq washed from I20 ;

Line3: Taq washed from I50 ;

Line4: Taq washed from I300 .

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'''Result'''

Taq from I20 and I50 have activity,and supertanant also.

Taq from I20 has high purity and quantity.

So, collect the Taq from I20 to do concerntration.

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[[Image:Taq 377 PCR for activity 1-12-2012-1.JPG]]

Date:01-13-2012 through 01-17-2012;

Page:003493-94

Line 1: Positive control;

Line 2: Taq eluted with I20;

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Date:01-24-2012

[[Image:Taq 377 PCR --measure Unit-1-25-2012.JPG|242x217px|Taq 377 PCR --measure Unit-1-25-2012.JPG]]

 

Experimenter: Yingying Lu

 Line1: Taq after concentration:

Line2: 1:2 Taq diluted with storage buffer;

Line3: 1:5

Line4: 1:10

Line5: 5U/ul 

Line6: NEB One Taq 5U/ul

Line7: SF Taq 2U/ul

 

[[Image:Taq 377 PCR --measure Unit 2-1-25-2012.JPG|239x241px|Taq 377 PCR --measure Unit 2-1-25-2012.JPG]] Notebook: Ruili Su 2

Page:003501

Experimenter: Ruili Su

Line1: 1:5 diluted taq with storage buffer; 

Line2: 1:25 diluted taq

Line3: 1:53 diluted taq

Line4: 1:54 diluted taq

Line5: 1:55 diluted taq

Line6: 1:100 diluted taq

Line7: Invitrogen Taq 5U/ul

Line8: NEB One Taq 5U/ul

 

 

 

 

 

 

 

 

 

 

 

 

 

 

[[Category:Protein_Purification]]

File:Taq 377 PCR --measure Unit 2-1-25-2012.JPG

2012-01-25T22:13:20Z

Ruili.su:

File:537 2nd digest 1-25-2012.JPG

2012-01-25T18:47:09Z

Ruili.su: