1719-pT7A3-MBP-(E3C)-NRF2
Cloning of 1719-pT7A3-MBP-(E3C)-NRF2
[edit]Purpose: Cloning of 1719-pT7A3-MBP-(E3C)-NRF2
Date: 2013-4-18---2013-4--22
Notebook:0
Page:
Clone ID:1719
Experimenters: yingying
Results
[edit]cloning
[edit]
lane5:pcr product
lane6:1699 pT7A3-MBP-(E3C)
enzyme:Nde1+Xho1
Colony Screening
[edit]Date: 2013-4-8
Make miniprep and digest with Nde1+Xho1
Which clone is used for making glycerol stock?
clone3 for stock
Result intepretation/Summary
[edit]- <br>
Conclusion
[edit]protein screening
[edit]
| lane |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
9 |
10 |
| clone ID | 626,pT7A3-Taq_d289 |
1717, pT7A3-6H-(TEV)-MBP-(3C) |
1719, pT7A3-MBP-(E3C)-NRF2 |
1720,pT7A3-MBP-(E3C)-Keap1 |
1719,
pT7A3-MBP-(E3C)-NRF2, fraction 38 |
1719, pT7A3-MBP-(E3C)-NRF2, fraction 23 |
cre, 3.9ug/ul |
cre,4/2/2013 |
1624,pCold-6His-GST-(E)-(3C) |
|
| host |
Nico21DE3plysN |
Nico21DE3plysN |
BL21DE3pRare2pLysS |
BL21DE3pRare2pLysS |
BL21DE3pRare2pLysS |
BL21DE3pRare2pLysS |
Nico21DE3plysN |
|||
| induction |
0.2 mM IPTG 25 oC o/n |
0.2 mM IPTG 25 oC o/n |
0.2 mM IPTG 25 oC o/n |
0.2 mM IPTG 25 oC o/n |
0.2 mM IPTG 25 oC o/n |
0.2 mM IPTG 25 oC o/n |
0.2 mM IPTG 25 oC o/n |
|||
| purification |
Ni beads |
Ni beads |
Ni beads |
Ni beads |
5ml HisTrap HP |
5ml HisTrap HP |
Ni beads |
|||
| MW |
61KD/5.97 |
49KD/5.46 |
112KD/4.82 |
115KD/5.57 |
112KD/4.82 |
112KD/4.82 |
33KD/6.0 |
Cloning of 1719-pT7A3-MBP-(E3C)-NRF2
[edit]Purpose: Cloning of 1719-pT7A3-MBP-(E3C)-NRF2
Date: 2013-7-10---2013-7-11
Notebook:006026
Page:006202
Clone ID:1719
Experimenters: yingying
Results
[edit]cloning
[edit]
PCR NRF2 from 1622 pLX304-NRF2
NRF2-Nde1-F
NRF2-Xho1-R
lane1: pcr product
lane3: vector 1721 pT7A3-MBP-(E3C)-SOD
enzymes: Nde1+Xho1
digest for 1hour and 30mins,then ligate
screening
[edit]2013-7-11
Nde1+Xho1
Cloning of 1719-pT7A3-MBP-(E3C)-NRF2
[edit]Purpose: Cloning of 1719-pT7A3-MBP-(E3C)-NRF2
Date: 2013-7-11---2013-
Notebook:006026
Page:006202
Clone ID:1719
Experimenters: yingying
Results
[edit]cloning
[edit]
PCR NRF2 from 1622 pLX304-NRF2
NRF2-Nde1-F
NRF2-Xho1-R
lane3: pcr product
lane4: vector 1699pT7A3-MBP-(E3C)
enzymes: 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 |