FAQ: Why do I see no product or low yield on an agarose gel after a PCR using Phusion® High-Fidelity DNA Polymerase?

  • Be sure to use an annealing temperature determined by the NEB Tm calculator.
  • Use fresh high-quality nucleotides. It is important to avoid dNTP mixes that contain 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.
  • The DNA template may be GC rich, or supercoiled.

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