4.8.1. Primer length, melting temperature and composition
Both amplification primers should ideally be the same length (around 20 nucleotides) with similar melting temperature (Tm) between 55oC-60oC, giving enough room for experimental annealing temperature optimization and long enough to avoid non-specific amplifications. 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. The primer sequences should be evenly balanced between A/T and G/C nucleotides and avoid long homopolymeric stretches (i.e. runs of more than 4 of the same nucleotide).