Protocol Time Savings
Amplicon: 500 bp, number of cycles: 30
| PCR Step | High Performance PCR (2-step) | Conventional PCR with Taq (3-step) |
|---|---|---|
| Total ramping time | 10.5 min 1 | 29 min |
| Total denaturation time | 40 s (10 s + 30 x 1 s) 2 | 8.5 min (1 min + 30 x 15 s) |
| Total annealing time | 0 s 3 | 15 min (30 x 30 s) |
| Total extension time | 4.5 min (30 x 7 s + 1 min) 4 | 15 min (30 x 20 s + 5 min) |
| Total protocol time | 16 min |
67.5 min |
1 Speed is due to the fast ramping rate (>8°C/s) of Piko Thermal Cyclers and minimal thermal resistance of UTW tubes.
2 Speed is due to several factors: Fast settling time (less than 1 s) of Piko Thermal Cyclers, low thermal resistance of UTW tubes, and the extreme stability of Phusion DNA Polymerase at high temperatures (98°C); shorter denaturation step is required than at 94°C.
3 Speed is due to the fast settling time of Piko Thermal Cycler and low thermal resistance of UTW tubes. In addition, a 2-step protocol is more often applicable because higher annealing temperatures are used with Phusion DNA Polymerases.
4 Speed is due to the high processivity of Phusion DNA Polymerases, which enables the polymerase to extend the strand faster than other DNA polymerases.
Total cycling times
Comparison of total cycling times in amplification of genomic DNA fragments between 0.7 - 2.0kb. The increased processivity of Phusion DNA Polymerases allows extremely short cycling times. Additionally, annealing temperatures used are 5°– 8°C above conventional PCR. Both of these shave valuable seconds off of each cycle and make 2-step protocols easily achievable. The extraordinary short hold times are due to the temperature stabilizing effect of the Piko Thermal Cyclers and UTW vessels, minimizing the time required to achieve the target temperature.