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  • FAQ: Why do I see no product or low yield on an agarose gel after a PCR using Phusion® Hot Start Flex DNA Polymerase?

    • Be sure to use an annealing temperature determined by the nearest neighbor method (or Tm Calculator ).
    • Use fresh high-quality nucleotides. It is important to avoid dNTP mixes that contains dUTP.
    • Use more template. Sample concentration may be too low.
    • Template DNA may be damaged. Use carefully purified template.
    • Lengthen extension time.
    • Increase cycle number.
    • Optimize enzyme concentration.
    • Denaturation temperature may be too low. Optimal denaturation temperature for most templates is 98°C or higher.