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Trouble-shooting: No product

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 NaCl concentration above 50 mM

Suggestion: Reduce NaCl concentration

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KCl concentration above 50 mM

Suggestion: Reduce KCl concentration

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