1465 pT7A3-MBP-3C
Cloning of 1465 pT7A3-MBP-3C
[edit]Purpose: Cloning of 1465 pT7A3-MBP-3C, native 3C, not E coli optimized
Date: 2012-11-21-- Notebook:0
Page:
Clone ID:1465
Experimenters: SHANYI
Results
[edit]cloning
[edit]
subclone from 1420 using Nde1 + Xho1 to 1429
Colony Screening
[edit]Date: 2012-11-
digest with Nde1 + Xho1
Protein Expression
[edit]2/18/2013
| Lane |
Clone ID |
Name |
Gene Name |
Host |
Induction | Purification |
Note |
| M |
Invitrogen |
||||||
| 1 |
1465 |
pT7A3-MBP-3C |
MBP-Native 3C |
3 ml |
Ni, |
Only MBP Expressed | |
| 2 |
1661 |
pT7A3-MBP-(3c)-NRF2 |
MBP-NRF2 |
3 ml, pRare2 |
Ni |
||
| 3 |
1661 |
pT7A3-MBP-(3c)-NRF2 |
MBP-NRF2 |
3 ml, pRare2PLysS |
Ni |
||
| 4-8 |
1613 |
pET28-cold-GST-3C |
GST-Native 3C |
8L |
After Ni and Glutathione column |
aggregate after sitting at 4 oC for 7 days, probably due to oxidation, need to add b-ME | |
| 9-11 |
1613 |
pET28-cold-GST-3C |
GST-Native 3C |
8L |
After Ni |
extract protein from cell pellet of 4~8 (above) |
Note: 9~11: Add 300 ml of buffer A to cell pellets from previous week purification, homogenize, spin and load supernatant to Ni column. Wash with Buffer A and elute with Buffer B (300 mM Imidazole). Got a lot of protein from pellet.