1748-pCold-8His-MBP-NRF2-6His
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Cloning of 1748-pCold-8His-MBP-NRF2-6His
[edit]Purpose: Cloning of 1748-pCold-8His-MBP-NRF2-6His
Date: 2013-5-7--2013-5-10
Notebook:0
Page:
Clone ID:1748
Experimenters: yingying
Results
[edit]cloning
[edit]
Which one is vector? which one is NRF2 donor?
lane4: 1719 pT7A3-MBP-(E3C)-NRF2
lane5: 1611 pCold-MBP-(3C)
enzyme: Nde1 + Xho1
Colony Screening
[edit]Date: 2013-5-10
Make miniprep and digest with Nde1 + Xho1
Which clone ? #6?
clone6 for stock
Result intepretation/Summary
[edit]Conclusion
[edit]Protein Expression
[edit]2013-5-20
[edit]
| lane |
clone ID |
host |
induction |
purification |
MW/pI |
Note |
| 1-8 |
1748,pCold-8His-MBP-NRF2-6His |
BL21(DE3)pRare2pLysS |
gel filtration fractions |
NRF2 not expressed | ||
| 9 |
1676,pCold-MBP-(3C)-KEAP1 |
BL21(DE3)pRare2pLysS |
||||
| 10 |
1726,pCMV5-hIgG-Lc-PKARIIb |
293T |
||||
| 11 |
1708, pT7A3-HTNCre |
NiCO21(DE3)pLysN |
SP-FF,MEP, |
|||
| 12 | Fermentas Page-Ruler Plus |
[edit]
Cloning of 1748-pCold-8His-MBP-NRF2-6His
[edit]Purpose: Cloning of 1748-pCold-8His-MBP-NRF2-6His
Date: 2013-7-10--2013-
Notebook:0
Page:006201
Clone ID:1748
Experimenters: yingying
Results
[edit]cloning
[edit]
PCR NRF2 from 1622 pLX304-NRF2
NRF2-Nde1-F
NRF2-Xho1-R
lane2: pcr product
lane4:1611 pCold-MBP-(3C)
enzymes: Nde1+Xho1
screening
[edit]2013-7-11
Nde1+Xho1
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 |