If a sample requires >10 fmol of DNA, such as when performing targeted capture of nuclear DNA, we have found that the SYBR Green signal can be unreliable. As a benchmark for determining the number of cycles that should be used during PCR, we have found that samples requiring ~600 fmol of total input DNA should be stopped between cycles six and eight. Importantly, for every twofold increase or decrease in the amount of DNA used in the PCR, the reactions should be stopped one cycle earlier or later, respectively. When first setting up DS for a new experimental system or genomic target, we recommend testing the number of cycles needed to maximize yield while avoiding higher-molecular-weight products by pausing the thermocycler every two cycles and carefully removing a small aliquot from the reaction. The aliquots can then be quantified on an Agilent TapeStation 2200 or Bioanalyzer 2100 to determine the optimal cycle number (Fig. 6a,b).
