Template:SJTU-BioX-Shanghai/Notebook/Construction/w10
Construction
Sep. 7
Transformation of PcpcG2-HR-Mut into DH5a and plating. After incubation for 12h, Inoculated single colonies into liquid LB culture (10 tubes for test group and 2 for control group).
Sep. 8
Plasmid extraction.
Double restriction enzyme digestion.
| Restriction Enzyme Digestion | |
|---|---|
| ddwater | 280μl |
| FD buffer | 40μl |
| XbaI | 20μl |
| EcoRI | 20μl |
| Template | 2μl/tube |
| Total volume | 400μl(20μl*20) |
- Template included PcpcG2-HR-Mut, PcpcG2-HR and 0 control.
Agarose gel electrophoresis test.
Sep. 9
Sequencing PcpcG2-HR-Mut-4 and PcpcG2-HR-Mut-9.
Sep. 10
Sequencing results proved right.
Sep. 17
Functional fragments restriction enzyme digestion.
| Pdark-GFP | Pdark-HR | PcpcG2-HR | |
|---|---|---|---|
| ddwater | 15μl | 15μl | 15μl |
| FD buffer | 2μl | 2μl | 2μl |
| XbaI | 1μl | 1μl | 1μl |
| PstI | 1μl | 1μl | 1μl |
| Template | 1.3μl | 1μl | 0.9μl |
| Total volume | 20μl | 20μl | 20μl |
