I recently tried to PCR amplify some sodium bisulfite converted DNA samples for bisulfite pyrosequencing. I am using two sets of published primers and they produced a single band for me in the past (one is 143bp and other is 341bp). Not sure if the primers are getting old or if the DNA is not getting fully converted. The primers are about a year old and stocks have been stored at -20C. I was able to get the one set of primers to work by lowering the annealing temperature from 55 to 51C. However, the other set of primers will not produce a single band. They worked at 59C but now only produce a smear with annealing temperatures from 62-49.
What is the problem?