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ion Insufficient heating during the denaturation step is a common cause of failure in a PCR reaction. It is very important that the reaction reaches a temperature at which complete strand separation occurs. A temperature of about 94C for 2 minutes should be adequate in most cases. As soon as the sample reaches 94C, it can be cooled to the annealing temperature. Extensive denaturation is probably unnecessary and limited exposure to elevated temperatures helps maintaining maximum polymerase activity throughout the reaction. DNA reaction buffers with higher Mg-concentration (4-5 mM) may require a higher denaturation temperature to allow complete separation of the DNA template strands. It is recommended to use the supplied buffer without adding further magnesium. 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
  • Closed circular DNA should be cleaved before the PCR, uncut circles renaturate too fast
Back to Top Machine based error Suggestions:
  • Calibrate the heating block and confirm actual block temperature
  • Run a diagnostic program for the particular machine - contact the machine manufacturer for specifics
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