PCR Inhibition from Contaminated Template

Symptom

No or weak PCR amplification despite correct reaction setup, with template DNA containing residual contaminants from extraction or purification that inhibit polymerase activity.

1. 1 Presence of PCR inhibitors (ethanol, phenol, salts, EDTA, SDS, heparin) in template preparation
2. 2 Template degradation visible as smearing after Mg²⁺ incubation
3. 3 Low 260/280 ratio (<1.8) indicating protein or phenol contamination
4. 4 Excessive template sample volume introducing inhibitors into reaction

1. 1 Further purify template by alcohol precipitation to remove salts and small molecules
2. 2 Perform drop dialysis against TE buffer to remove inhibitors
3. 3 Use Monarch® Spin PCR & DNA Cleanup Kit (5 µg) (NEB #T1130) for efficient purification
4. 4 Decrease template sample volume in reaction while maintaining DNA amount
5. 5 Analyze DNA by gel electrophoresis before and after Mg²⁺ incubation to assess integrity
6. 6 Start with fresh template preparation using high-quality extraction kits

Related Video (2)

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