PCR Inhibition from Contaminated Template (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

No or weak PCR amplification despite correct reaction setup, with template DNA containing residual contaminants from extraction or purification that inhibit polymerase activity. Common causes: 1) Presence of PCR inhibitors (ethanol, phenol, salts, EDTA, SDS, heparin) in template preparation 2) Template degradation visible as smearing after Mg²⁺ incubation 3) Low 260/280 ratio (<1.8) indicating protein or phenol contamination 4) Excessive template sample volume introducing inhibitors into reaction Solutions: 1) Further purify template by alcohol precipitation to remove salts and small molecules 2) Perform drop dialysis against TE buffer to remove inhibitors 3) Use Monarch® Spin PCR & DNA Cleanup Kit (5 µg) (NEB #T1130) for efficient purification 4) Decrease template sample volume in reaction while maintaining DNA amount 5) Analyze DNA by gel electrophoresis before and after Mg²⁺ incubation to assess integrity 6) Start with fresh template preparation using high-quality extraction kits