Cycling times and temperatures have a direct impact on yield and primer binding for accuracy of results. We have rigorously tested the cycling parameters of the PCR reaction to optimize the time and temperatures with the primer design and master mix formulation. We do not recommend varying the number of cycles or the cycling temperatures.
MP and AP control DNA was amplified for increasing cycle times. We found that 35 cycles produces the most robust bands while reducing primer dimer formation.
Positive controls were amplified with increased annealing temperature. We found that 58.0 °C gave the most robust bands without generating non-specific amplification.