PCR + Primers
Primer design and PCR optimization are critical for the success of many eDNA studies. I’ve been having many problems with a few primer sets I’ve designed. They are currently amplifying off taxa targets. When I look back at the reference database sequences, there are multiple basepair mismatches in both the forward and reverse primers. I can’t figure out why it is amplifying; it is unbelievable. I guess you won’t know how primers react until you test them on real-life samples. The primer design software I use is AlleleID which Pete Countway has in his lab. The system can search for primer binding regions on specific taxa while excluding closely related species. This makes designing species-specific assays reasonably straightforward. It took us under 5 minutes from start to finish to design our SAR-CoV2 primers, which we used for our in-class demonstration. Speaking of, below are the result of the Biomeme run from class.
