I have a question about experimental design about one experiment that I am currently involved.
After DNA extraction we applied a PCR protocol using primers for 3 regions (two ITS region, and 16S). within all the regions we had 5 primes F and 5 primers R: consisting of an Illumina adaptor stub, a variable length region of 1-5 N (used as a code), and then the primer.
We performed a AMPure bead cleaning to remove prime dimers.
For each samples we have three different amplicons set: two ITS and one 16S.
What we see on the gel is two prominent bands: one in the 380-400 bp range (expected), and another at 80 bp which we can't determine what that is.
Our concern is that in an illumina run the 80 bp will occupy all the signal.
My PI is concerned about the 80 bp band, and we want to know why it happened and if we can filter this out somehow. Does anyone have an idea about what happened and if it is possible to filter it out?
Thank you so much for your help!