The number of PCR cycles is an important variable. Additional cycles beyond the exponential phase can result in the appearance of nonspecific, higher-molecular-weight products (Fig. 6a,b). To avoid this complication, we carefully monitor the number of PCR cycles. For samples requiring <10 fmol of input DNA, such as for mtDNA, we monitor the reaction by qPCR and remove the reactions one or two cycles before the SYBR Green signal plateaus, which typically occurs between cycles 15 and 20. Notably, the specific plateau point will be dependent on the amount of DNA used in the PCR and should be carefully monitored.
