Asymmetric PCR for ssDNA Template Generation v1

This protocol describes a specialized PCR approach to generate high-purity single-stranded DNA (ssDNA) templates, ranging from 120 to 1000 bp, for use in enzymatic activity assays. Unlike standard PCR, which uses equal concentrations of primers to produce double-stranded DNA (dsDNA), this method employs an unequal primer ratio—typically 50:1—to favor the linear amplification of a single strand once the limiting primer is exhausted. The protocol is optimized for high-fidelity polymerases like Q5, though it is adaptable to other systems provided the unequal primer concentrations and extended cycling (80 cycles) are maintained to ensure the depletion of the limiting primer. Final product quality is verified via gel electrophoresis to ensure the absence of dsDNA contamination, which can otherwise lead to inaccurate downstream measurements.