Paul.xu at 15:21, 13 August 2013

2013-08-13T15:21:34Z

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== '''Cloning of 1683-pCold-MBP-(3C)-NRF2''' ==

'''Purpose:''' Cloning of 1683-pCold-MBP-(3C)-NRF2

'''Date: '''2013-2-21---2013-2-25

'''Notebook:0'''

'''Page:'''

'''Clone ID:1683'''

'''Experimenters:''' yingying

== '''Results''' ==

=== subcloning ===

[[Image:1683 clone 2-22-2013.JPG]] 

subclone from 1661 pT7A3-MBP-(3c)-NRF2 to 1611 pCold-MBP-(3C) using Nde1 + Xho1 Cloned By 

 

 

=== Colony Screening ===

Date: 2013-2-25

Make miniprep and digest with  Nde1 + Xho1

[[Image:1683 digest detect 2-25-2013.JPG]] 

 

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

== '''Conclusion''' ==

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

== ==

== '''Cloning of 1683-pCold-MBP-(3C)-NRF2''' ==

'''Purpose:''' Redo Cloning of 1683-pCold-MBP-(3C)-NRF2, 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:1683'''

'''Experimenters:''' yingying

== '''Results''' ==

PCR 

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

=== Colony Screening ===

Date: 2013-3-3

Make miniprep and digest with  

1. Nde1 + Xho1.

[[Image:1661 1674 1683 Nde1 Xho1.jpg]] 

2. HindIII + Pst1 

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

 

 

== Protein Expression ==

2013/3/5 

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

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

clone 1 

|
4 ml, 

BL21(DE3)Star, pRare2-pLysS

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

Clone 2 

|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

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

BL21(DE3)Star, pRare2-pLysS 

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

BL21(DE3)Star, pRare2-pLysS 

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

BL21(DE3)Star, pRare2-pLysS 

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

== ==

== 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]] 
| 
|}

 

 

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

== '''Conclusion''' ==

[[Category:Cloning]]

1683-pCold-MBP-(3C)-NRF2 - Revision history

Paul.xu at 15:21, 13 August 2013