Annealing temperature

To amplify products with high fidelity it is important to choose an annealing temperature that is optimal for the specific combination of primers and template sequence. A carefully chosen annealing temperature (Ta) will significantly affect product yield and reduce the amplification of partially polymerized products. Ta calculations used by SeqBuilder Pro are as presented in Rychlik et al. :

Ta = 0.3 Tm Primer + 0.7 Tm Product – 14.9

Where Tm Primer is the minimum Tm of the two primers using the appropriate method and Tm Product is the product Tm using the appropriate GC content formula.