I want to amplify RAPD and ISSR markers using primers which has 27-37 0C of annealing temperatures. I'm conducting this study for a plant evolutionary study. I've tried several times with Solis Biodyne, 5 X FIREPol Master mix and it didn't give results ( didn't successfully amplified the genes) as the recommended annealing temperature scale there is 54-66 0C . I've done several optimization steps and I was end with no results. Therefore I thought of changing the PCR master mixture and the brand for the lower annealing temperature PCR.
I expect all polymerases to work at low temperatures ( just slower) but would avoid hot start enzymes. Try the cheapest ordinary taq available but also check the OD260/280 of the template dna and look closely at the primer history. If these are dissolved in water not TE and are old and inherited from other workers then have them made fresh in case they are degraded. It is possible that at low temperatures the dna template has a lot of secondary structure so it might be worth running the pcr in 1M betaine or 6% DMSO to lessen the secondary structure
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