HomeFAQsWhy 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?
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.
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