1661-pT7A3-MBP-(3c)-NRF2
Cloning of 1661-pT7A3-MBP-(3c)-NRF2
[edit]Purpose: Cloning of 1661-pT7A3-MBP-(3c)-NRF2
Date: 2013-2--11----2013-2-14
Notebook:0
Page:
Clone ID:1661
Experimenters: yingying
Results
[edit]subcloning
[edit]
| band 5 |
band 6 | |
| 1626, pET28-cold-NRF2 |
1514,pT7A3-MBP-(3C) | |
| enzymes |
Nde1 + Xho1. | |
Colony Screening
[edit]Date: 2013-2-14
Make miniprep and digest with Nde1 + Xho1.
Result intepretation/Summary
[edit]Conclusion
[edit]Not good. Forward primer has an extra G, will not express
Cloning of 1661-pT7A3-MBP-(3c)-NRF2
[edit]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
[edit]PCR
Colony Screening
[edit]Date: 2013-3-3
Make miniprep and digest with
1. Nde1 + Xho1.
2. HindIII + Pst1
Protein Expression
[edit]2013/3/5
| 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
[edit]| 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 |