A primer on PCR

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.

  1. Dilute the template
  2. Decrease the specifity of the PCR program
  3. Increase the amount of MasterMix
  4. Increase the size of the reaction
  5. Re-extract template DNA

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