Editing 1661-pT7A3-MBP-(3c)-NRF2

== '''Cloning of 1661-pT7A3-MBP-(3c)-NRF2'''  ==

'''Purpose:''' Cloning of 1661-pT7A3-MBP-(3c)-NRF2 

'''Date: '''2013-2--11----2013-2-14 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:1661''' 

'''Experimenters:''' yingying 

== '''Results'''  ==

=== subcloning  ===

[[Image:554+560+1661 clone 2-11-2013.JPG]]<br> 

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
|-
| <br> 
| band 5<br> 
| band 6<br>
|-
| <br> 
| 1626, pET28-cold-NRF2<br> 
| 1514,pT7A3-MBP-(3C)<br>
|-
| enzymes<br> 
| colspan="2" | &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Nde1 + Xho1.<br>
|}

<br> 

{| cellspacing="1" cellpadding="1" border="1"
|-
| <br>
|}

=== Colony Screening  ===

Date: 2013-2-14 

Make miniprep and digest with&nbsp; Nde1 + Xho1. 

[[Image:1661 digest detect 2-14-2013.JPG]] 

<br> 

<br> 

<br> 

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

*<br>

== '''Conclusion'''  ==

Not good. Forward primer has an extra G, will not&nbsp; express 

==  ==

== '''Cloning of 1661-pT7A3-MBP-(3c)-NRF2'''  ==

'''Purpose:''' Redo Cloning of 1661-pT7A3-MBP-(3c)-NRF, previous cloning did not produce expression clone due to bad design of F primer. 

'''Date: '''2013-3-2---2013-3- 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:1661''' 

'''Experimenters:''' yingying 

== '''Results'''  ==

PCR<br> 

{| cellspacing="1" cellpadding="1" border="1"
|-
| <br>
|}

=== Colony Screening  ===

Date: 2013-3-3 

Make miniprep and digest with&nbsp; <br> 

1. Nde1 + Xho1. 

[[Image:1661 1674 1683 Nde1 Xho1.jpg]]<br> 

2. HindIII + Pst1<br> 

[[Image:1661 1674 1683 HindIII Pst1.JPG]] 

<br> 

<br> 

== Protein Expression  ==

2013/3/5<br> 

[[Image:1661 1674 1683 1676 3 6 2013.jpg]]<br> 

{| cellspacing="1" cellpadding="1" border="1" width="684"
|-
| Lane<br> 
| Name<br> 
| Clone ID<br> 
| Host<br> 
| Induction<br> 
| Purification<br>
|-
| M<br> 
| Fermentas PageRuler Plus<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 1<br> 
| pT7A3-MBP-(3c)-NRF2 <br> 
| 
1661 

clone 1<br> 

| 
4 ml,<br> 

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 2<br> 
| pT7A3-MBP-(3c)-KEAP1<br> 
| 
1661 

Clone 2<br> 

| 
4 ml, 

BL21(DE3)Star, pRare2-pLysS <br> 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 3<br> 
| pCold-GST-(3c)-NRF2<br> 
| 1674<br> 
| 
4 ml, 

BL21(DE3)Star, pRare2-pLysS <br> 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads (wrong, should use Glutathione agarose beads, no his tag)<br>
|-
| 4<br> 
| pCold-GST-(3c)-NRF2<br> 
| 1674<br> 
| 
4 ml, 

BL21(DE3)Star, pRare2-pLysS <br> 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads (wrong, should use Glutathione agarose beads, no his tag)<br>
|-
| 5<br> 
| pCold-MBP-(3C)-NRF2<br> 
| 1683<br> 
| 
4 ml, 

BL21(DE3)Star, pRare2-pLysS <br> 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 6<br> 
| pCold-MBP-(3C)-NRF2<br> 
| 1683<br> 
| 4 ml,BL21(DE3)Star, pRare2-pLysS <br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 7<br> 
| pT7A3-MBP-(3c)-KEAP1<br> 
| 1625<br> 
| 4 ml,BL21(DE3)Star, pRare2-pLysS <br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 8<br> 
| pCold-MBP-(3C)-KEAP1<br> 
| 1676<br> 
| 4 ml,BL21(DE3)Star, pRare2-pLysS <br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 9 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 10 
| Purified MBP 
| <br> 
| <br> 
| <br> 
| <br>
|}

<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>

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

== '''Conclusion'''  ==

[[Category:Cloning]]