I have a primer set. Tm of Forward primer is 56.6 and that of Reverse primer is 63.5. Which common temperature I should use for PCR reaction to gett a good amplification?
You should choose the annealing temperature based on the oligo that has the lower melting temperature. However, it is generally recommended to design primers such that their melting temperatures differ by no more than 5 oC. See for example the Tm calculator available from the Thermo Fisher web site.
you have to modify your primers to obtain a good match regarding the melting temperatures. Often an addition or substraction of one nucleotide is enough.
If you already had the primers synthesized, analyze them in any PCR primer analysis software. That should give you a Tm that you can use as a starting point, or issue a warning regarding disparities in primer Tm, etc. Also, if you have access to a gradient machine you can set up several reactions across the block with different Tm to select the best performer under your specific conditions.
Hope it helps!
Agree with Pierre Béguin
You should choose an annealing temp according to the primer having the lower Tm. First you can make a 10uL reaction using this Tm, in most of the cases PCR works fine around the ~55C annealing temp. If it does not work, then you can try a gradient PCR with a range of annealing temp from 55C to 65C.
I hope it will help. All the best
I start to use a gradient PCR which comprises both the Tm. After in function of the result I select the correct Tm.
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