1752-pCold-TF-(E3C)-NRF2
Cloning of 1752-pCold-TF-(E3C)-NRF2
[edit]Purpose: Cloning of 1752-pCold-TF-(E3C)-NRF2
Date: 2013-5-24----2013-5-28
Notebook:0
Page:
Clone ID:1752
Experimenters: yingying
Results
[edit]cloning
[edit]
subclone from 1748 pCold-8His-MBP-NRF2-6His or 1719 pT7A3-MBP-(E3C)-NRF2 into 1614 pCold-TF-(3c) using Nde1 + Xho1
cloning screening
[edit]2013-5-28
Make miniprep and digest with Nde1 + Xho1
Cloning of 1752-pCold-TF-(E3C)-NRF2
[edit]Purpose: Cloning of 1752-pCold-TF-(E3C)-NRF2
Date: 2013-5-28----2013-5-30
Notebook:0
Page:
Clone ID:1752
Experimenters: yingying
cloning
[edit]
1: 1719pT7A3-MBP-(E3C)-NRF2
2:1729 pCold-TF-(E3C)-3C
3:1748 pCold-8His-MBP-NRF2-6His
cloning screening
[edit]2013-5-30
Make miniprep and digest with Nde1 + Xho1 /Hind111+Pst1
5/27/2013
[edit]Purpose: Cloning of 1752-pCold-TF-(E3C)-NRF2
Date: 2013-5-27----2013-5-28
Notebook:0
Page:
Clone ID:1752
Experimenters: paul
Digest vector (left) 1729 pCold-TF-(E3C)-3C with Nde1 + Xho1
Right: Digest Nef2 in pT7A3-MBP-(E3C), clone1719 pT7A3-MBP-(E3C)-NRF2 with Nde1 + Xho1
cut out vector and NRF2, purify and ligate, transform NEB Turbo
Colony Screening
[edit]Date: 2013-5-28
Make miniprep and digest with nde1+xho1/hind111+pst1
2013-5-29
above clones digest with different enzymes
clone 1:Nde1+xho1
clone 3:Nco1+xho1
clone 4:EcoR1+xho1
clone 5:Hind111+xho1
Result intepretation/Summary
[edit]Conclusion
[edit]Cloning of 1752-pCold-TF-(E3C)-NRF2
[edit]Purpose: Cloning of 1752-pCold-TF-(E3C)-NRF2
Date: 2013-5
Notebook:0
Page:
Clone ID:1752
Experimenters: shanyi
Results
[edit]cloning
[edit]
subclone from 1748 pCold-8His-MBP-NRF2-6His or 1719 pT7A3-MBP-(E3C)-NRF2 into 1729using Nde1 + Xho1
cloning screening
[edit]2013-5-30
Make miniprep and digest with Nde1 + Xho1
Protein Expression & Purification
[edit]June, 2013:
Expression in BL21(DE3)pRare2 pLysS, 5 L, purify on 10 ml Ni column. Concentrate using spin concentrator, then load to Biosil TSK-Gel 250
1 & 2: the first high MW peak
3, 4, 5, 6: the second low MW peak.
These bands are not right size.
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 |