FAQ: Why are there low molecular weight discrete bands on an agarose gel after a PCR using Phusion® Hot Start Flex 2X Master Mix?

Non-specific products are being amplified. To avoid this we suggest the following:
  • Raise the annealing temperature.
  • Shorten extension time.
  • Optimize Mg2+ concentration.
  • Titrate template amount.
  • Lower primer concentration.
  • Re-calculate primer Tms using NEB's Tm Calculator .