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Trouble-shooting: Misincorporation or low fidelity

nucleotides Back to Top The pH of the reaction buffer is too high A pH 8.3 is optimal Suggestions:
  1. pH of reaction buffer can be lowered to 5-6
  2. Base substitution changes decreased by 60 fold and frame-shift changes decreased by 11 fold with a 3 unit pH decrease
Back to Top Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers Suggestions:
  • Increase initial denaturation temperature to 95-97C
  • Denature DNA minus enzyme & buffer for 4-6 minutes
  • Increase cycling denaturation time 15-30 seconds
  • Try "Hot Start" technique
  • Add T4 Gene 32 protein 3-5 l/ml
  • When designing primers, make sure there is no more than 2 bases of homology at the 3' end. Use a primer design program if available.
  • Consider addition of cosolvent to reaction buffer:
  • 3-15% DMSO
  • 1-10% Formamide
  • 5-15% Polyethylene glycol
  • 10-15% glycerol
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