Basic Phusion PCR protocol¶
PCR recipe¶
| Component | Volume | Final Concentration |
|---|---|---|
| 5X Phusion HF buffer | 10 | 1X |
| 2 mM* dNTPs | 5 | 200 μM |
| Forward primer, 10 μM | 2.5 | 500 nM |
| Reverse primer, 10 μM | 2.5 | 500 nM |
| Plasmid template, 1 ng/μL | 1 | 20 pg/μL |
| Homemade Phusion | 0.5 | |
| Water | 28.5 |
*Lab stocks of 10 mM dNTPs can be diluted 1:5 in water.
Thermal cycling¶
| Time | Temperature |
|---|---|
| 1:00 | 98 °C |
| 0:30 | 98 °C |
| 0:30 | 60 °C* |
| 0:30 + 0:15/kb | 72 °C |
| x30 cycles | |
| 5:00 | 72 °C |
| 0:01 | 22 °C** |
*Adjust to suit the Tm of the primers, if necessary. You can calculate optimal Tm at NEB's website here.
**It is better for the thermal cycler to end PCR at room temperature. Extended holds at 4 °C decrease the lifespan of the block. PCRs are stable when kept at room temperature overnight!
Considerations to increase chances of successful amplification¶
- Difficult templates might benefit from changing HF buffer to GC buffer. Adding magnesium or DMSO can also sometimes help with difficult templates.
- PCR may not work with too much or too little template; it is a good idea to test several concentrations.
- Amplifying genomic DNA or cDNA may require significantly more template. Amplifying cDNA also seems to work better when cDNA is made with oligo dT, rather than random hexamers.
- When using homemade Phusion, set up PCRs on ice and add PCRs to a hot thermal cycler block (put a 98 °C "hold" step at the beginning of your protocol, and press "skip step" once PCRs are added).
- Homemade Phusion does not always perform at the advertised extension time of 4 kb/minute. Err on the side of longer extension times for best results.