I am trying to get a 150-200 bp PCR product from genomic DNA (for pyrosequencing). However, after trying different annealing temperatures (52-62 C), extension times (30-60 s), MgCl concentrations (2-3), primer concentrations and 2 different TAQs, I still don't get any visible PCR products on a 1.6% agarose gel. I have a positive control (1200 bp), which always works perfectly. With 2 primers flanking my region of interest, I got a PCR product which I sequenced and which seems to be correct.
Has anyone ever experienced something like that and any further advice what I could do about this?
Thank you so much!!