PCR/qPCR Contamination & NTC Amplification Triage

Pain point

NTC/negative controls amplify or show melt peaks, invalidating runs and spreading contamination risk.

Repro/diagnostic steps

1. Record which controls are positive (NTC only? extraction blanks too?).
2. Confirm product type: melt curve shape and/or gel band size vs expected.
3. Map workflow zones: where master mix is made, where template is added, where amplicons are opened.

Root causes (common)

• Carryover from prior PCR products (amplicon aerosol).
• Cross-contamination via pipettes, tips, shared reagents, shared PPE.
• Workflow fails to separate pre- and post-amplification areas.

Fix workflow

1. Enforce physical separation of pre- vs post-amplification areas; dedicated pipettes/consumables.
2. Rotate in fresh aliquots; replace reagents one-by-one to identify the source.
3. Consider UNG/UDG carryover prevention if compatible with assay.
4. Add routine negative controls (NTC + extraction blank) into every run until stable.

Expected result

• NTC and extraction blanks remain clean across runs; positives amplify as expected.

References

• https://www.thermofisher.com/tw/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/good-laboratory-practice-avoid-contamination-qpcr-experiments.html
• https://www.thermofisher.com/io/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/amplification-no-template-control.html
• https://www.idtdna.com/page/support-and-education/decoded-plus/could-your-pcr-be-affected-by-contamination