I am trying to perform a simple PCR reaction to linearize a plasmid (so that I can insert a gene by Gibson assembly), but it is driving me mad by failing over and over again. The plasmid is pcDNA3.1, and I have two PCR primers, designed to prime in opposite directions from my insertion site, so if it works I SHOULD get linearized plasmid!
But so far, if I use 25-30 cycles of amplification, I get several new bands of DNA on an agarose gel, both bigger and smaller than my template. Far more bands than I expect. But if I use less cycles, as in 18 cycles I get nothing. The gel is just blank. No PCR product whatsoever. What I NEED TO see is a PCR product that migrates more slowly than my template (linearized plasmid), and nothing else. But I either get far too many products of all sizes, or nothing.
Does anyone have any idea what I am doing wrong? I double checked my primers and see nothing wrong with how they are designed. I am at a loss.
Tufts University Boston