Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
Previously working assay fails completely when switching to different master mix brand; positive controls fail; original master mix works but new one does not despite similar specifications
Variable and incorrect standard curve efficiency when using serial dilutions; effect more pronounced when same dilution series stored at 4°C and reused; inconsistent differences between amplification plots; problem resolves when different operator uses different tubes
PCR efficiency calculated from standard curve is greater than 120%; ΔCq between 10-fold dilutions is much less than expected 3.3 cycles (e.g., 1.5 cycles); standard curve gradient indicates abnormally high efficiency
Low concentration data points do not fit linear standard curve profile; NTC shows amplification with lower Tm and broader melt peak than positive samples; primer dimers visible on gel, inversely proportional to template concentration
Low or absent fluorescence in both test sample and positive control; correct PCR product is visible on gel; one probe in multiplex shows consistently high background with no amplification signal; background fluorescence equivalent to water control
Positive control amplifies successfully but test sample known to contain target shows no amplification; undiluted template fails while dilutions may show improved amplification
Amplification plots are clearly abnormal with sections dipping below zero dR; data cannot be used as presented; plots appear distorted
Very low Cq values for concentrated samples; amplification plots are not regularly spaced and appear abnormal; background fluorescence is significantly higher; minimal fluorescence yield through the reaction
Cq data for standard curve dilutions are irregularly spaced; ΔCq between dilutions is inconsistent and decreases with increasing dilutions; replicates are precise but pattern is abnormal
Assay is insensitive and amplification plots look abnormal with pronounced drift of the baseline; fluorescence signals are weak or irregular
ReadyMix produces amplification in standard PCR but completely fails in real-time qPCR applications; equivalent products from other suppliers work well
JumpStart Taq ReadyMix does not work as well as similar products from different suppliers; amplification is weak or absent despite correct assay design
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