1682-pCold-6His-GST-(E)-(3C)-NRF2
Cloning of 1682-pCold-6His-GST-(E)-(3C)-NRF2
[edit]Purpose: Cloning of 1682-pCold-6His-GST-(E)-(3C)-NRF2
Date: 2013-6-6------2013-6-7
Notebook:0
Page:
Clone ID:1682
Experimenters: yingying
Results
[edit]subcloning
[edit]1
subclone from 1661 pT7A3-MBP-(3c)-NRF2 to 1624 pCold-6His-GST-(E)-(3C) Nde1 + Xho1<--by Yingying
6/7/2013: Digested with Nde1 + Xho1 (done by Yingying)
the following steps were done by Paul
1: 1661 pT7A3-MBP-(3c)-NRF2
2: vector 1624 pCold-6His-GST-(E)-(3C)
gel purification (Qiagen column), elute with 20 ul water.
ligation, 2.5 ul T4 ligase buffer with 1 ul of T4 and 1 ul T7 DNA ligase, 1 hour, at room temperature
transform 100 ul NEB Turbo, recovery time 1 hour
Colony Screening
[edit]Date: 2013-6-7
Make miniprep and digest with Nde1 + Xho1<-- By yingying
Comment by Paul: This gel shows some of the subcloning steps were carelessly done, or were not done properly, most of time this could be caused by rush to do experiment, hoping to "finish" experiment sooner so you can go home erarly or catch bus on time.
The following steps were done by Paul
6/8/2013
pick 10 colonies, grow in LB/Kan with 5% glycerol and 2.5% glucose. make minprep, digest with Nde1 + Kpn1
all clonies are positive, clone #8 vector size is a little bit smaller, do not know why.
digest colone 1 with three pair of enzymes:
1: Nde1 + Xho1: good
2. Nhe1 + Kpn1: good
3. EcoR1 + Pst1: good
Transform BL21Star (DE3)/pRare2pLysS with 0.5 ul of plasmid. Grow o/n and pick a few colonies to grow in 4 L of LB/K/C for protein expression.
Make glycerol stock of clone #1 in NEB Turbo
Make glycerol stock of clone # in BL21Star (DE3)/pRare2pLysS
Result intepretation/Summary
[edit]Conclusion
[edit]Protein Expression and Purification
[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 |