Boosting PCR accuracy – tips for maximizing amplification fidelity

Speaking of Mol Bio

In this Mol Bio Minutes episode, Laurynas Alijošius shares a personal story that every molecular biologist can relate to—running PCR, cloning, and sequencing, only to discover frustrating errors in the DNA.

This episode dives deep into PCR accuracy and why it matters for everything from sequencing to cloning and long-read library prep. Laurynas breaks down the major contributors to PCR error, including the fidelity of DNA polymerase, primer design flaws, template impurities, and suboptimal cycling conditions. He then offers a range of solutions—like switching to high-fidelity enzymes, using ready-to-go master mixes, and optimizing magnesium ion concentrations. He also explains how reducing cycle numbers and fine-tuning annealing temperatures can minimize unwanted amplification and ensure more reliable data.

Whether you're troubleshooting or proactively optimizing your workflow, this episode is packed with tips and tools to help you increase PCR accuracy, reduce costs, and save time. Episode notes contain links to enzyme comparisons, primer design tools, and cycling guides to help you PCR with precision.

Helpful resource links mentioned in this episode:

• Thermo Scientific web tools for primer design and analysis, and more
• The PCR Learning Center with lots of helpful tips and information
• Learn more about PCR reagents and enzymes
• Browse and purchase PCR enzymes and master mixes
• Access the PCR troubleshooting guide
• Download the molecular biology handbook

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Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology.

For Research Use Only. Not for use in diagnostic procedures.