If difference between Tm of forward and reverse SSR primers is more than 10 C and this primer is useful then what to do for getting good result?

11 November 2016 1 3K Report

My question is that....If difference between Tm (Melting temperature) of forward and reverse SSR primers is more than 10 0C and this primer is useful then what to do for getting good result?

Robert M Givens

Primer annealing is an equilibrium process, so one may use the higher of the two temperatures to assure proper stringency but compensate for the reduced stability of the lower Tm primer by raising its concentration. Try steps of 2-fold, 5-fold and 10-fold excess of the lower Tm primer over the "normal" amount per reaction of the higher Tm primer. One may also combine this "concentration compensation" titration approach with trying various intermediate annealing temperatures to optimize between yield and stringency.

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