I've been having some trouble amplifying my bisulfite treated DNA with the primers I have. I've been using Zymo's EZ Methylation Gold kit for conversion. I had some success with some universally methylated and non-methylated pUC-19 controls from Zymo, but the primers I've designed aren't amplifying anything. I know some things that I plan on altering, like ordering a hot start enzyme (we tried to get by with a taq polymerase since we already had it ready), altering my PCR protocol, and using nested primers, but I had a few technical questions about it.
Why is nested PCR recommended if larger bisulfite PCR products are difficult to amplify? According to recommendations, the new primers I designed have 1st products no bigger than 500 bp, then my semi-nested or nested product are no bigger than 225 bp. The primers are anywhere from 24-32 bp, typically don't contain polystretches more than 5 of any base, have a GC clamp, and only use a degenerate base for a CpG when absolutely necessary.
My PI thinks that an "old fashioned" purification with proteinase K and ethanol will work to purify my PCR product before sending in, is that sufficient?
Are the Tms in methyl primer express trustworthy? Because I've seen an extension stage recommended on this forum for about 64C, but that is close to the Tms of some of the primers I'm designing so I could see that creating a problem.
Is getting a high fidelity enzyme that reads through uracil recommended? Like Thermo Scientific's Phusion U Hot Start? I've seen some say that they've had great results with that, but I know that conditions in some enzymes have been optimized for bisulfite PCR, like ZymoTaq.
Sorry for so many questions! I appreciate any help! This forum has been great and has provided me with so much useful information.