FAQ: Why are there high molecular weight smears or DNA in the wells of an agarose gel after a PCR using Phusion® Hot Start Flex 2X Master Mix?

Strong protein binding to the DNA may be preventing proper gel separation. Try adding 1% SDS to the loading dye just before running the gel. Or non-specific products are being amplified. To avoid this we suggest the following:
  • Shorten extension time.
  • Reduce total number of cycles.
  • Increase annealing temperature or try 2-step protocol.
  • Optimize Mg2+ concentration.
  • Lower primer concentration.