Template:SJTU-BioX-Shanghai/Notebook/Construction/w9
Contents
Construction
Aug. 31
- Plasmid extraction.
- Double restriction enzyme digestion test for Pdark-GFP, Pdark-HR and PcpcG2-HR.
| Restriction Enzyme Digestion | |
|---|---|
| ddwater | 360μl |
| FD buffer | 450μl |
| XbaI | 45μl |
| PstI | 45μl |
| Template | 1μl/tube |
| Total volume | 900μl(20μl*39) |
Agarose gel extraction test.
Sep. 2
Gel anlysis of the restriction enzyme digestion product for a second time.
Sep. 3
Sequencing samples. Pdark-GFP-3, Pdark-GFP-8, PcpcG2-HR-4,PcpcG2-HR-6, Pdark-HR-2, Pdark-HR-4.
Sep. 6
All sequenced sample were successful and right. Site-directed mutation on PcpcG2-HR to eliminate the EcoRI sites via PCR method.
| PcpcG2-HR-Mut | |
| ddwater | 105μl |
| G2Mut-F | 10μl (10μM) |
| G2Mut-R | 10μl (10μM) |
| PcpcG2-HR(pSB1C3) | 2μl |
| PrimeStar Mix | 125μl |
| Total volume | 250μl（25μl*8）( |
| 98℃ 3min | |
| 98℃ 10s | ×32 cycle |
| 65℃ 5s | |
| 72℃ 30s | |
| 72℃ 5min | |
| 12℃ ∞ |
DpnI digestion for 2h. Electrophoresis test and gel extraction afterwards.
Homologous recombination of PcpcG2-HR-Mut.
| PG2-Mut | Control-1 | |
|---|---|---|
| ddwater | 12μl | 14μl |
| 5x CEII Buffer | 4μl | 4μl |
| PCR product | 2μl | 2μl |
| Exnase II | 2μl | 0μl |
37℃ for 30min. Product storaged in -20℃ overnight.