9.3.3. Assay optimization

Once a PCR assay has been designed, it should be optimized experimentally for annealing temperature (using annealing temperature gradients), primer concentration and cycling times (see the BEEBOOK paper on molecular techniques; Evans et al., 2013). Optimization usually identifies the highest annealing temperature, the lowest primer concentrations and the shortest incubation time that consistently generates the right product, without artefacts, at a consistent amplification cycle (see section 12: “Quality control”).