I've been working on this gene (wolbachia gene) for nearly half a year now and over these months, I've been having problems with my wsp primers. some days it'll be as good as anything, and some sucks.
We have tried changing the polymerase from Taq pol to Phusion high fidelity polymerase. it was working for a while and now it's gone chaos again.
I have attached a photo of my latest screening. the last two lanes are positive control and negative control consecutively.
I was wondering if anyone could give me an idea as to why there're more than one bands and why are they so many bands at the bottom. I shouldn't be seeing any other bands after ~560bp (expected band size).
FYI, DNA were extracted only 2 days prior to this screening, all reagents have been freshly made for troubleshooting.
thanks in advance!