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Trouble-shooting: Misincorporation or low fidelity

Here are some troubleshooting hints that we have gathered regarding misincorporation or low fidelity of PCR reactions: Magnesium concentration too high Nucleotide concentration is too high or unbalanced The pH of the reaction buffer is too high Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers Damaged template DNA Non-optimal Mg++ concentration

Suggestion:Titrate magnesium concentration using our PCR Optimization Kit.

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Nucleotide concentration is too high or unbalanced The standard concentration is 20-200 M of each nucleotide Suggestions:
  1. Check the concentration of stock solutions of all nucleotides
  2. Double check the final concentrations of all 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
Back to Top Damaged template DNA Suggestions:
  1. Minimize cycle number, since repeated heating/cooling at high pH can damage template (Most templates require 25-35 cycles)
  2. Check integrity of template DNA
  3. Reduce cycling denature time to 15 - 30 seconds
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