A qPCR genotyping assay is returning no Cq value at all, or the no-template control is amplifying with a broad low-temperature melt peak instead of a clean single peak, making it impossible to trust the genotype calls.

The hack / solution

For no amplification: verify polymerase, dNTPs, and both primers were actually added to the master mix — a reaction missing any one of those will never amplify, however clean the template is. 2) Quantify the DNA template on a fluorometer instead of assuming its concentration, and confirm it falls inside the assay's validated input range before repeating the run. 3) For primer-dimers, recognizable as a melt peak below about 75°C, well under the intended amplicon peak: switch primer concentration down from 500 nM to 100-200 nM, then raise the annealing temperature 1-2°C at a time until the dimer peak clears. 4) If dimers persist after retuning concentration and annealing temperature, redesign the primers under stricter dimer-formation criteria, or switch to a probe-based chemistry instead of SYBR-based detection.

📎Source: Azure Biosystems — qPCR Troubleshooting: No Amplification & Primer-Dimers🔗View source🗓️Added: 2026-07-16✅Reviewed by: MouseStuff Admin