1779 pTCold-MBP-(3C-TEV)-NRF2
Cloning of 1779 pTCold-MBP-(3C-TEV)-NRF2
[edit]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
[edit]subcloning
[edit]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
Colony Screening
[edit]Date: 6/30/2013
miniprep digest with Pst1 or Kpn1
these results suggest that the subcloning did not work.
Result intepretation/Summary
[edit]Conclusion
[edit]Cloning of 1779 pTCold-MBP-(3C-TEV)-NRF2
[edit]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
[edit]subcloning
[edit]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
Colony Screening
[edit]Date: 6/30/2013
miniprep digest with Kpn1
Result intepretation/Summary
[edit]Conclusion
[edit]protein screening
[edit]2013-7-5
| 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
[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 |