Paul.xu at 15:30, 13 August 2013

2013-08-13T15:30:39Z

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== '''Cloning of 1779 pTCold-MBP-(3C-TEV)-NRF2''' ==

'''Purpose:''' subcloning of  NRF2 into pTCold-MBP-(3C-TEV) to generate  pTCold-MBP-(3C-TEV)-NRF2 

'''Date: '''2013-6-30~

'''Notebook:'''  

'''Page:'''    

'''Clone ID: 1779'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== subcloning ===

Date; 6/30/2013

digest vector 1677 pTCold-MBP-(3C) with Nhe1 + Xho1 for 2 hours, digest 1683 pCold-MBP-(3C)-NRF2, 1769 pET28b-6His-NRF2 with Nhe1 + Xho1 

[[Image:Vector1677-Nhe1 Xho1.JPG]]    [[Image:DIGEST FOR1779.JPG]]

=== Colony Screening ===

Date: 6/30/2013 

miniprep digest with Pst1 or Kpn1

[[Image:1779 Kpn1 6 31 2013.JPG]]

[[Image:1779 Pst1 6 31 2013.JPG]]

these results suggest that the subcloning did not work.

 

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

== '''Conclusion''' ==

== '''Cloning of 1779 pTCold-MBP-(3C-TEV)-NRF2''' ==

'''Purpose:''' subcloning of  NRF2 into pTCold-MBP-(3C-TEV) to generate  pTCold-MBP-(3C-TEV)-NRF2 

'''Date: '''2013-7-1~

'''Notebook:'''  

'''Page:'''    

'''Clone ID: 1779'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== subcloning ===

Date; 7/1/2013

digest vector 1677 pTCold-MBP-(3C) with Nhe1 + Xho1 for 2 hours,

digest 1683 pCold-MBP-(3C)-NRF2, 1674 pCold-GST-(3c)-NRF2,1698pT7A3-NRF2with Nhe1 + Xho1 

[[Image:Digest for 1779 1619.JPG]]

   

 

=== Colony Screening ===

Date: 6/30/2013 

miniprep digest with Kpn1

 

 

 

[[Image:1779DIGEST.JPG]]

 

 

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

== '''Conclusion''' ==

== '''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]]

1779 pTCold-MBP-(3C-TEV)-NRF2 - Revision history

Paul.xu at 15:30, 13 August 2013