Routine PCR

The Polymerase Chain Reaction (PCR) is a rapid method for amplifying defined target DNA sequences, typically within a heterogeneous population of DNA sequences. Routine PCR refers to the amplification of a single DNA target typically ≤ 5kb having a GC content between ~ 40-60% using standard conditions and reagents. Routine PCR is a major component of many molecular biology workflows and is generally fast, robust and reproducible.

Routine PCR is typically performed with Taq or a Taq-based blend of polymerases. that provides enhanced performance. These blends often include a proofreading polymerase that provides enhanced performance and moderately higher fidelity (e.g., 2X) than Taq alone. In cases where true high-fidelity is required, a robust proofreading polymerase (i.e., Q5®) should be used instead of a Taq blend.

For best PCR results, please follow the recommendations that come with each enzyme and calculate an annealing temperature for each primer set using the NEB Tm calculator. For most Routine PCR experiments, limited optimizations are required to achieve reproducible and robust results.

  1. 5 Tips for Setting Up Your PCR

    Experiencing amplification frustration? Follow Melanie’s 5 quick and easy tips for PCR setup to improve your yields.

  2. How Can I Improve Amplification of GC-rich Regions

    The secondary structures that form within GC-rich regions can make efficient DNA amplification difficult. Nicole’s suggestions can help!

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FAQs for Routine PCR

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