Primer annealing temperature, length and composition

Both amplification primers should have similar melting temperature (Tm), length and composition. It is useful to design all assays and primers around the same annealing temperature, so that a single cycling program can be used for all assays, and that different assays can be run concurrently with the same program, on the same plate. 56oC is a good, standard, robust target for the in silico estimated Tm for primers.