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Protocol: PCR
1X Mastermix 20μl reaction, add in this order
| Reagent | Volume [μl] |
|---|---|
| Water | Complete to total volume of 20μl |
| HF-Buffer (5x) | 4 |
| DMSO (optional) | 0.6 |
| dNTPs | 0.4 |
| Forward primer (50μM) | 0.2 |
| Reverse primer (50μM) | 0.2 |
| Template (10ng/μl) | 0.5 |
| Phusion HF polymerase | 0.2 |
Multiply everything by the number of reactions you want to do +1. Take the HF-Buffer, DMSO and dNTPs out the of the freezer in advance so it all has time to thaw. Take the Phusion-HF polymerase out of the freezer only when adding it and put it right back again. If the reactions have different primers and/or template, add the polymerase right after the dNTPs, slit the mastermix and add the rest.
Tm: The primers must have similar Tms. Always chose the lower one. If not sure about the Tm, perform a gradient PCR to find out.
