Table 2.
Touchdown Polymerase Chain Reaction Temperature Cycling Parameters
| Phase 1 | Step | Temp | Time |
|---|---|---|---|
| 1 | Initial denature | 98°C | 3°min |
| Stop and add 2.5 μL Phusion polymerase per tube | |||
| Phase 2 | Step | Temp | Time |
|---|---|---|---|
| 2 | Initial denature | 98°C | 20 s |
| 3 | Denature | 98°C | 10 s |
| 4 | TD anneal | 70–55°C | 30 s |
| 5 | Elongation | 72°C | 3′45″ |
| Steps 3–5 repeated 15 times, temp decreasing 1°C/cycle | |||
| Phase 3 | Step | Temp | Time |
|---|---|---|---|
| 6 | Denature | 98°C | 10 s |
| 7 | Anneal | 58°C | 30 s |
| 8 | Elongation | 72°C | 3′45″ |
| Steps 6–8 repeated 33 times | |||
| Termination | Step | Temp | Time |
|---|---|---|---|
| 9 | Elongation | 72°C | 7 min |
| 10 | Halt reaction | 11°C | 10 min |
The Phusion DNA polymerase is added after the initial 3°min denaturation step. In the touchdown phase is the annealing temperature is set 12–15°C above the theoretical annealing temperature of the primers and lowered by 1°C for 15 cycles. After this the annealing temperature is set to 58–60°C.