I have been trying to use PCR to amplify cDNA using standard Taq polymerase. I am able to amplify beta actin, but not my target. Unfortunately, the Tm for my two primers is somewhat far apart (one is 57 and the other is 62). Based on the NEB calculator, I chose to start with annealing temperature of 52. After this failed, I tried to use the same annealing temp with more cycles, increased amount of template (1-4ul from a 20ul RT reaction), and increased primer concentration (200nM-600nM). Neither of these steps fixed the problem. Finally, I performed an annealing gradient from 50-60 degrees ... when the products were run on gel, it turned out ONLY a 55 degree annealing temp gave a band, which was not particularly strong. I have seen gels from annealing gradients that where the product appears to increase and then peaks at the optimal temp - is it also possible for the reaction to ONLY work at a specific annealing temp. Also, will increasing annealing time help at all? I am a novice at PCR so any advice will help!

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