Editing 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His

== '''Cloning of 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His'''  ==

'''Purpose:''' Cloning of 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His 

'''Date: '''2013-7-16--- 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:1796''' 

'''Experimenters:''' yingying 

== '''Results'''  ==

=== cloning  ===

subclone MBP-NRF2 fragment from 1779 pTCold-MBP-(3C-TEV)-NRF2 to 1719 pT7A3-MBP-(E3C)-NRF2 using Bsa1 + EcoR1<br> 

<br> 

[[Image:1796clone1 7-16-2013.jpg]]<br> 

{| cellspacing="1" cellpadding="1" border="1" width="366"
|-
| lane<br> 
| 1 insert<br> 
| 2 vector<br>
|-
| <br> 
| 1779 pTCold-MBP-(3C-TEV)-NRF2<br> 
| 1719 pT7A3-MBP-(E3C)-NRF2<br>
|-
| <br> 
| colspan="2" | &nbsp;&nbsp; EcoR1<br>
|}

<br> 

lanes above&nbsp; do gel extraction and digest again with Bsa1 at 50C for 1hour and do ligation<br> 

[[Image:1796clone2 7-16-2013.jpg]]<br> 

{| cellspacing="1" cellpadding="1" border="1" width="350"
|-
| lane<br> 
| 1 insert<br> 
| 2 vector<br>
|-
| <br> 
| 1779 pTCold-MBP-(3C-TEV)-NRF2<br> 
| 1719 pT7A3-MBP-(E3C)-NRF2<br>
|-
| enzymes<br> 
| colspan="2" | &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Bsa1<br>
|}

<br> 

<br> 

<br> 

=== Colony Screening  ===

Date: 2013-7-17 

Make miniprep and digest with Pst1 

[[Image:1796digest1 7-17-2013.JPG]] 

<br> 

Date: 2013-7-18 

Make miniprep and digest with Xho1 + EcoR1<br> 

[[Image:1796 digest2 7-18-2013.JPG]] 

<br> 

<br> 

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

== '''Conclusion'''  ==

<br> 

== protein expression result of NRF2 clones <br>  ==

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

<br> 

<br> 

[[Category:Cloning]]