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High Performance PCR - General Protocol Design
High Performance PCR is as simple as any PCR, but it follows slightly modified rules for creating the protocol. Most of these changes can be found within the Phusion DNA Polymerases instruction sheets. The key points to remember include:
- Raising the denaturing temperature to 98°C.
- Raising the annealing temperature by up to 8°C (Tm +3°C, > 20 nt) as compared with a conventional protocol.
- Shortening the denaturation, annealing and extension steps.
Piko Thermal Cyclers provide fast ramping rates and ultra-fast settling times (less than 1 s with UTW vessels). These features enable the denaturation and annealing steps to be kept extremely short, which further speeds up the PCR protocol. When making all of these changes, sub-30 minute PCR protocols, and often less than 20 minute protocols are achieved. In addition, because the annealing step is performed at higher temperatures than with other polymerases, a 2-step protocol is easier to achieve. This furthermore shortens the total protocol time.
Cycling instructions using Phusion® Flash High-Fidelity PCR Master Mix
Cycle step |
2-step protocol |
3-step protocol |
Cycles | ||
|---|---|---|---|---|---|
Temp |
Time |
Temp |
Time |
||
Initial denatuation |
98°C |
10 s |
98°C |
10 s |
1 |
Denaturation |
98°C |
0 or 1 s |
98°C | 0 or 1 s |
30
|
Annealing1 |
- |
- |
X°C | 5 s | |
Extension |
72°C |
15 s/1 kb |
72°C |
15 s/1 kb |
|
Final extension |
72°C |
1 min |
72°C |
1 min |
1 |
|
4 °C |
hold |
4 °C |
hold |
|
1A two-step PCR protocol is applicable with primers whose Tm values are, when calculated with Finnzymes' Tm calculator, at least 69°C or 72°C (primers >20 nt or ≤20 nt, respectively). Primers >20 nt: Anneal at Tm +3°C of the lower Tm primer. Primers ≤20 nt: Use an annealing temperature equal to the Tm of the lower Tm primer.
