Basic techniques in Molecular biology
Polymerase chain reaction
| Total volume | 50/μL |
|---|
| DDW | 36 |
| 10×Buffer | 5 |
| dNTP（2.5mM） | 5 |
| FP（10μM） | 1 |
| RP（10μM） | 1 |
| DNA template | 1 |
| EasyPfu DNA Polymerase | 1 |
Thermocycling
The PCRmachine should be set to run the following steps
| Pre-denaturation | 94℃ | 5min |
|---|
| transsexual | 94℃ | 30s | 30circles |
| annealing | Tm-5℃ | 30s |
| extend | 72℃ | 0.5kb/min |
| extend | 72℃ | 5min |
extraction plasmid
1-4 mL of the bacterial solution is centrifuged at 12,000 rpm for 2 min, and the bacterial solution is discarded.
2) Resuspend by adding 200μL SolutionI, then add 400μL SolutionII and invert upside and down slowly until the bacterial solution is transparent. Next add 300μL SolutionIII and mix. Further, 300 ul of chloroform is added and mixed, and centrifuge at 13,000 rpm for 2 minutes. 700 ul of the supernatant is removed into a new 1.5 mL tube, and add 490 μL of 70% isopropanol.Next centrifuge at 13,000 rpm for 2 minutes, and the supernatant is discarded. After rinsing the precipitate twice with 500 μL of 75% ethanol, centrifuges at 12,000 rpm for 2 min.Finally,dry at 65℃ and dissolve with 15-20 mL of sterile water previously heated.
PCR Purfication and gel extraction
PCR Purfication
