PCR
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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 |