Secondary PCR bands

Hi all!

We’ve been doing PCR again lately with the Caporaso primers. For many samples we have several other bands on the gel, mainly one major extra band that is larger than the expected one. It is no problem to cut out the wanted product from the gel after multiplexing, and most people seem to do that anyway. However that distorts concentrations when using the Sequal plates, as the extra products bind to that also and are only removed in the gel purification step after.
Excuse the horrible image:

Do you also have those extra bands? Do you ignore them or did you optimize PCR conditions to get rid of them, and if yes, how?

Any idea what those bands are? Side products, or just secondary structure variations of the main product?


I’ve seen larger bands in samples that likely have lots of eukaryal host DNA relative to bacterial DNA. Since they seem to be sample type dependent, I haven’t been able to compare similar samples with and without the extra band to see if there’s a big difference. I sequence them as is