Paul.xu at 15:25, 13 August 2013

2013-08-13T15:25:11Z

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== '''Cloning of 1752-pCold-TF-(E3C)-NRF2''' ==

'''Purpose:''' Cloning of 1752-pCold-TF-(E3C)-NRF2

'''Date: '''2013-5-24----2013-5-28

'''Notebook:0'''

'''Page:'''

'''Clone ID:1752'''

'''Experimenters:''' yingying

== '''Results''' ==

== ==

=== cloning ===

[[Image:1752 clone 5-24-2013.jpg]]

 

subclone from 1748 pCold-8His-MBP-NRF2-6His or 1719 pT7A3-MBP-(E3C)-NRF2 into 1614 pCold-TF-(3c) using Nde1 + Xho1

=== cloning screening ===

2013-5-28 

Make miniprep and digest with Nde1 + Xho1 

[[Image:1752digest 5-28-2013.jpg]]

 

= Cloning of 1752-pCold-TF-(E3C)-NRF2 =

Purpose: Cloning of 1752-pCold-TF-(E3C)-NRF2

Date: 2013-5-28----2013-5-30

Notebook:0

Page:

Clone ID:1752

Experimenters: yingying 

=== cloning ===

[[Image:1752 clone 5-28-2013.JPG]] 

1: 1719pT7A3-MBP-(E3C)-NRF2 

2:1729 pCold-TF-(E3C)-3C 

3:1748 pCold-8His-MBP-NRF2-6His 

=== cloning screening ===

2013-5-30

Make miniprep and digest with Nde1 + Xho1 /Hind111+Pst1 

[[Image:1752 digest 5-30-2013.JPG]] 

 

 

=== 5/27/2013 ===

Purpose: Cloning of 1752-pCold-TF-(E3C)-NRF2

Date: 2013-5-27----2013-5-28

Notebook:0

Page:

Clone ID:1752

Experimenters: paul 

Digest vector (left) 1729 pCold-TF-(E3C)-3C with Nde1 + Xho1 

Right: Digest Nef2 in pT7A3-MBP-(E3C), clone1719  pT7A3-MBP-(E3C)-NRF2 with Nde1 + Xho1 

cut out vector and NRF2, purify and ligate, transform NEB Turbo

== '''[[Image:1752 2013 5 27.JPG]]''' ==

=== Colony Screening ===

Date: 2013-5-28

Make miniprep and digest with nde1+xho1/hind111+pst1 

[[Image:1752digestp 5-28-2013.JPG]] 

 

2013-5-29 

above clones digest with different enzymes 

[[Image:1752 digest4 5-29-2013.JPG]] 

clone 1:Nde1+xho1 

clone 3:Nco1+xho1 

clone 4:EcoR1+xho1 

clone 5:Hind111+xho1 

 

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

== '''Conclusion''' ==

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

== '''Cloning of 1752-pCold-TF-(E3C)-NRF2''' ==

'''Purpose:''' Cloning of 1752-pCold-TF-(E3C)-NRF2

'''Date: '''2013-5

'''Notebook:0'''

'''Page:'''

'''Clone ID:1752'''

'''Experimenters:''' shanyi

== '''Results''' ==

== ==

=== cloning ===

[[Image:Digest for 1752.JPG]] 

 

subclone from 1748 pCold-8His-MBP-NRF2-6His or 1719 pT7A3-MBP-(E3C)-NRF2 into 1729using Nde1 + Xho1

=== cloning screening ===

2013-5-30 

Make miniprep and digest with Nde1 + Xho1 

[[Image:1752digest.JPG]][[Image:1752digest2.JPG]] 

== Protein Expression & Purification ==

June, 2013: 

Expression in BL21(DE3)pRare2 pLysS, 5 L, purify on 10 ml Ni column. Concentrate using spin concentrator, then load to Biosil TSK-Gel 250 

 

== [[Image:1752 gel filtration purification.jpg]] ==

1 & 2: the first high MW peak

3, 4, 5, 6: the second low MW peak. 

These bands are not right size. 

 

== protein screening ==

2013-7-5

[[Image:1661+1682+1683+1719+1748+1752+1771+1779protein screening 7-5-2013.JPG]]

{| cellspacing="1" cellpadding="1" border="1" width="200"
|-
| Lane 
| Name 
| Host 
| Induction 
| Purification 
| MW 
| Conclusion 
|-
| 1 
| 1661,pT7A3-MBP-(3c)-NRF2 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 112KD 
| yes 
|-
| 2 
| 1682, pCold-6His-GST-(E)-(3C)-NRF2 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 97KD 
| yes 
|-
| 3 
| 1683,pCold-MBP-(3C)-NRF2 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 113KD 
| yes 
|-
| 4 
| 1719,T7A3-MBP-(E3C)-NRF2 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 112KD 
| no 
|-
| 5 
| 1748,pCold-8His-MBP-NRF2-6His 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 120KD 
| no 
|-
| 6 
| 1752,pCold-TF-(E3C)-NRF2 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 120KD 
| yes 
|-
| 7 
| 1771,pT7A3-MBP-(3C)-PCK1 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 110KD 
| yes 
|-
| 8 
| 1779, pTCold-MBP-(3C-TEV)-NRF2 
| 4 ml,
NiCO21(DE3), pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 113KD 
| yes 
|-
| 9 
| 1779, pTCold-MBP-(3C-TEV)-NRF2 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 113KD 
| yes 
|}

 

== protein expression result of NRF2 clones ==

{| cellspacing="1" cellpadding="1" border="1" width="569"
|-
| clone ID 
| expression 
| gel reslut 
| 
|-
| 1626, pET28-cold-NRF2 
| no 
| [[1625 1626 1656]] 
| 
|-
| 1661,pT7A3-MBP-(3c)-NRF2 
| yes, 
| [[1661 1674 1683]] 
| 
|-
| 1674,pCold-GST-(3c)-NRF2 
| need to use GST beads 
| [[1661 1674 1683]] 
| 
|-
| 1682,pCold-6His-GST-(E)-(3C)-NRF2 
| yes 
| [[1661 1682 1683 1719 1748 1752 1771 1779]] 
| 
|-
| 1683,pCold-MBP-(3C)-NRF2 
| yes, 
| [[1661 1674 1683]] 
| 
|-
| 1698,pT7A3-NRF2 
| no 
| [[1676 1759 1698 1769 1747 1769 1757 1682p]] 
| 
|-
| 1719,pT7A3-MBP-(E3C)-NRF2 
| yes 
| [[631 1497 1542 1719 1748 1765 1780 1781 1782 1797]] 
| 
|-
| 1748,pCold-8His-MBP-NRF2-6His 
| yes 
| [[631 1497 1542 1719 1748 1765 1780 1781 1782 1797]] 
| 
|-
| 1752,pCold-TF-(E3C)-NRF2 
| yes 
| [[1661 1682 1683 1719 1748 1752 1771 1779]] 
| 
|-
| 1769,pET28b-6His-NRF2 
| no 
| [[1676 1759 1698 1769 1747 1769 1757 1682p]] 
| 
|-
| 1796,pT7A3-MBP-(3C-TEV)-NRF2-6His 
| not sure 
| [[1720 1757 1759 1796 1800 1803 1804 1809 1759 MBP-Keap1 1719]] 
| 
|-
| 1779,pTCold-MBP-(3C-TEV)-NRF2 
| yes 
| [[1661 1682 1683 1719 1748 1752 1771 1779]] 
| 
|}

 

 

[[Category:Cloning]]

1752-pCold-TF-(E3C)-NRF2 - Revision history

Paul.xu at 15:25, 13 August 2013