1796-pT7A3-MBP-(3C-TEV)-NRF2-6His
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Cloning of 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His
[edit]Purpose: Cloning of 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His
Date: 2013-7-16---
Notebook:0
Page:
Clone ID:1796
Experimenters: yingying
Results
[edit]cloning
[edit]subclone MBP-NRF2 fragment from 1779 pTCold-MBP-(3C-TEV)-NRF2 to 1719 pT7A3-MBP-(E3C)-NRF2 using Bsa1 + EcoR1
| lane |
1 insert |
2 vector |
| 1779 pTCold-MBP-(3C-TEV)-NRF2 |
1719 pT7A3-MBP-(E3C)-NRF2 | |
| EcoR1 | ||
lanes above do gel extraction and digest again with Bsa1 at 50C for 1hour and do ligation
| lane |
1 insert |
2 vector |
| 1779 pTCold-MBP-(3C-TEV)-NRF2 |
1719 pT7A3-MBP-(E3C)-NRF2 | |
| enzymes |
Bsa1 | |
Colony Screening
[edit]Date: 2013-7-17
Make miniprep and digest with Pst1
Date: 2013-7-18
Make miniprep and digest with Xho1 + EcoR1
Result intepretation/Summary
[edit]Conclusion
[edit]
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 |