Hi i am wrking with SSR markers. I calculate the anealing temp of primers by taking the average of Tm of FP and RP and deducting 5 to it. Keeping touch down PCR. Is it correct calculation
Most of the time, we used that calculation as a starting point to test the temperature to get the optimal annealing temperature. From that point, we could increase or decrease the temperature by 1 or 2 degree based on this initial findings. The best way is to test the annealing temperature by gradient PCR, just like Artur has suggested to you.
Go for gradient PCR with varying range of Ta for your primer set, Do gradient PCR in 8 pcr tubes with different annealing temperature for same product(DNA/cDNA). You may get a amplification in any one of the annealing temperature.