FAQ: Why do I see no product or low yield on an agarose gel after a PCR using Phusion® Hot Start Flex 2X Master Mix?

  • Be sure to use an annealing temperature determined by the nearest neighbor method (or Tm calculator).
  • Use more template. Sample concentration may be too low.
  • Template DNA may be damaged. Use carefully purified template.
  • Lengthen extension time.
  • Increase cycle number.
  • Denaturation temperature may be too low. Optimal denaturation temperature for most templates is 98°C or higher.