qPCR: ntc amplification

1. Ranked causes

   1. Amplicon or template contamination in reagents / waterFresh water + new master-mix lot clears NTC; old aliquots stay positive.
   2. Primer-dimer or non-specific products read as amplification (SYBR)Melt shows low-Tm peak; probe assay on same primers is clean.
   3. Threshold / baseline setting flags noise as CtRaw curves are flat; auto-threshold placed in noise.
   4. Pipetting carry-over from standards into NTC wellsNTC Ct tracks nearby high-standard wells; re-plate with fresh tips clears it.

2. Recovery steps

   1. Replace water, primers, and master-mix aliquots; set up NTC firstRemoves the most common contamination reservoirs.
   2. Inspect melt curves; raise anneal temp or redesign if primer-dimer dominatesDistinguishes true contamination from SYBR primer-dimer signal.
   3. Re-check Ct with manual threshold above NTC noise floorAvoids calling flat NTCs as positive from auto-baseline artefacts.

3. Controls first

   • Does the NTC amplification show a melt peak matching the specific amplicon Tm?If yes: True contamination (or carry-over) — replace reagents and re-plate carefully.
   • Does the NTC amplification show a melt peak matching the specific amplicon Tm?If no: Likely primer-dimer or threshold artefact — adjust chemistry/analysis before assuming contamination.

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