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Team:University College London/Protocols/Phusion
From 2012.igem.org
Phusion High Fidelity Polymerase
| Component | 20 µl Reaction | 50 µl Reaction | Final Concentration |
|---|---|---|---|
| Nuclease-free water | to 20 µl | to 50 µl | |
| 5X Phusion HF or GC Buffer | 4 µl | 10 µl | 1X |
| 10 mM dNTPs | 0.4 µl | 1 µl | 200 µM |
| 10 µM Forward Primer | 1 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 250 ng |
| DMSO (optional) | (0.6 µl) | (1.5 µl) | 3% |
| Phusion DNA Polymerase | 0.2 µl | 0.5 µl | 1.0 units/50 µl PCR |
Thermocycling conditions for a routine PCR: Initial denaturation: 98°C 30 seconds
25–35 cycles: 98°C 5–10 seconds 45–72°C 10–30 seconds 72°C 15–30 seconds/kb
Final extension: 72°C 5–10 minutes
Hold: 4°C
