The PCR gods are a fickle sort and it’s an art to appease them. This strange intersection between science and the occult can be at the best of times trying, but fear not for I have braved the trials of PCR and write to offer advice.
To start I am sharing my standard PCR reaction. 10μL may seem like a tiny amount of product, but it’s enough to allow for testing on an agarose gel, amplicon cleanup for sequencing, and evaporation.
I use standard 10-μL PCR reactions, containing:
- 5μL of PCR MasterMix (Promega, Madison, Wisconsin, USA)
- 2.5μL of DNA-free H2O
- 0.5μL MgCl2
- 0.5μL forward primer
- 0.5μL reverse primer
- 1μL of template DNA
I then use touchdown PCR programs optimized for each primer pair to maximize the primer specificity. PCR product is then checked with Agarose gels. If the reaction failed I troubleshoot by trying each step below. Most of the time, diluting the template solves the problem.
- Dilute the template
- Decrease the specifity of the PCR program
- Increase the amount of MasterMix
- Increase the size of the reaction
- Re-extract template DNA