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3 years ago in Biology , Molecular Biology By Shreya K

How can large PCR fragments be amplified in molecular biology?

Working with genomic regions over 5kb, I consistently get weak or no yield with my standard Taq polymerase. I need reliable, bench-tested tips to improve fidelity and efficiency for these challenging amplifications.

PolymeraseChainReaction

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By Suresh Answered 2 years ago

From my lab work, amplifying large fragments requires a shift in mindset from routine PCR. First, I would recommend ditching standard Taq for a high-fidelity, processive enzyme blend like those designed specifically for long-range PCR. Critically, optimize your extension time allow at least 1 minute per kilobase. I have seen the biggest improvements come from adjusting buffer composition, often increasing Mg2+ and using additives like DMSO or betaine to ease strand separation and prevent secondary structures that halt the polymerase.

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