FAQ: Why does NEB recommend 40-45 cycles?

Amplification of 40 cycles is sufficient for high copy targets, or when using a well-characterized assay where non-template amplification is not a concern.

We suggest 45 cycles to maximize confidence in detecting low- to single-copy targets. Additional cycles may also help detect non-specific amplification for a given primer set when using dye-based qPCR mixes.