PCR
Jump to navigation
Jump to search
Protocol for Phusion
- Add the following volumes to a round-lidded PCR tube:
| Reagents |
Volume (µL) |
|---|---|
| ddH2O |
up to 50 µL |
| HF 5x buffer |
10 |
| ddNTPs |
1 |
| Primer A |
0.5 |
| Primer B |
0.5 |
| DNA |
X |
| DNA polymerase |
0.5-2 |
| DMSO (optional) |
(1.5) |
| Total |
50 |
Note: GC buffer can be used instead of HF buffer when using GC-rich templates
- Add the tube to the thermocycler and use the following times:
| Step | Temperature (°C) | Time (s) | Number of cycles |
|---|---|---|---|
|
Initial denaturation |
98 | 30 | 1 |
| Denaturation | 98 | 5-10 | 35-45 |
| Annealing |
>20nt; Tm + 3°C <20nt; Tm (lower of the two primers) |
10-30 (time depends on complexity) |
35-45 |
| Extension | 72 | 15-30/kb | 35-45 |
| Final extension | 72 | 5 min | 1 |
| Infinite hold | 4 | hold | 1 |