I designed some primers on MethPrimer to target my gene of interest. I have previously optimized primers for bisulfite converted DNA so this is the first time designing and optimizing them. Above 59C no product, 59-57 very faint band of correct size, and below 57 has very strong primer dimer bands. I have repeated the gradient with hot start taq polymerase and it doesn't help. Tried 40 and 45 cycles of PCR, and varying input amount of bisulfite converted DNA. The band is so weak that I am not able to excise for gel purification. I have a considerable amount of experience optimizing PCR conditions just do not know if the primer design is bad.
Any recommendations for enhancing product or trying to get rid of primer dimers? I have fought with primer dimers in the past but my band of interest was strong enough I could gel purify.
Any general recommendations for primer design?
Amplicon size? Mine is 700bp
Tm? These are 59 and 60C.