Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
PCR reaction fails to produce expected amplification or yields significantly reduced product, despite optimized reagents and cycling parameters. The issue traces to suboptimal thermal transfer or contamination from the plastic vessel itself.
Quantitative PCR shows inconsistent Cq values or fluorescence intensities between technical replicates in different wells, despite identical reaction setup. Well-to-well variability exceeds acceptable coefficient of variation.
PCR tubes become crushed, collapsed, or deformed after thermal cycling, potentially causing sample loss or compromised seal integrity. Tubes may show visible damage especially when using tube strips.
After PCR program completion, plates or tubes are found melted or stuck to the thermal cycler block, making removal difficult and potentially damaging samples. Plastic shows signs of thermal degradation.
Visible reduction in reaction volume after thermal cycling, with condensation on tube caps or film. May result in concentrated reagents, failed reactions, or inability to recover product.
Individual tube caps become dislodged during thermal cycling, exposing samples to evaporation and potential contamination. Caps are found loose or completely separated from tubes after run completion.
Quantitative PCR shows weak fluorescence signal across all wells, making accurate quantification difficult. Signal intensity is lower than expected despite adequate template and reagent quality.
Sealing film peels away from plate wells either before or during thermal cycling, leading to sample evaporation or cross-contamination. Film appears to have poor bonding with well rim surfaces.
No-template controls (NTC) or negative controls show unexpected amplification. Particularly problematic in sensitive applications like human identification qPCR where trace DNA contamination produces false positives.
New protocols and pitfalls, in your inbox
A short email when we add notable lab videos and failure cases. No spam, unsubscribe anytime.