PCR protocol (Phusion)
- Thaw PCR mastermix on ice for 10min.
- add 2µL of each primers (at 10 µM) in a PCR tube.
- add 1µL of the DNA (at 1 ng/µL) you want to amplify in the same tube.
- add 25µL of Life Technologies Phusion High-Fidelity PCR Master Mix with HF Buffer.
- add 20µL of DNase/RNase-free water to the tube.
- Thermocycling conditions
- initial denaturation 98°C for 30 seconds
- 35 cycles
- 98°C for 30 seconds
- 57°C for 30 seconds
- 72°C 30 seconds/kb
- final extension of 72°C for 10 minutes
- infinite hold 12°C
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