FAQ: I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® Hot Start Flex DNA Polymerase?

  • Use more template. Sample concentration may be too low.
  • Template DNA may be damaged. Use carefully purified template.
  • Optimize enzyme concentration by testing a titration of enzyme in the reaction (0.25-2 units/50μl reactions).
  • Increase number of cycles.
  • Lengthen extension time.
  • Re-calculate primer Tms using NEB's Tm Calculator .