Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
Gel electrophoresis shows no visible band at the expected product size after PCR amplification, indicating complete reaction failure.
Gel electrophoresis shows PCR product band(s) at unexpected molecular weight, either larger or smaller than the predicted amplicon size.
Gel electrophoresis reveals multiple bands, smearing, or bands at incorrect sizes in addition to or instead of the expected product, indicating lack of amplification specificity.
No or weak PCR product when amplifying GC-rich sequences (>65% GC content), long amplicons (>10 kb), or high-complexity genomic DNA despite optimization of standard parameters.
Unexpected products appear in negative controls or multiple samples show identical non-specific bands, indicating contamination from previous amplifications or environmental sources.
No or weak PCR amplification despite correct reaction setup, with template DNA containing residual contaminants from extraction or purification that inhibit polymerase activity.
Sequencing of the PCR product reveals mutations, insertions, deletions, or other sequence variations compared to the original template DNA.
Gel shows multiple bands instead of single target band, or primer-dimers appear as small molecular weight products. The desired product may be present but accompanied by unwanted amplification artifacts.
PCR fails or produces very weak amplification specifically with high GC content templates (>65%). Standard protocols work with other templates but not GC-rich sequences.
PCR amplification fails or produces very weak bands. Thermal cycling parameters appear correct, but reaction components may be improperly balanced or missing.
No PCR product is obtained despite correct thermal cycling and component concentrations. Investigation reveals potential primer-related issues including design flaws or contamination.
Multiple bands or primer-dimers appear on gel despite optimized thermal cycling. Component concentrations may be promoting nonspecific primer interactions or amplification.
Gel displays smeared bands suggesting degraded products or heterogeneous amplification. Template-related issues such as degradation, contamination, or excessive concentration are suspected.
Gel shows smeared or diffuse bands rather than discrete sharp bands. The smearing pattern suggests heterogeneous product populations from excessive amplification or nonspecific priming.
No visible PCR product band appears on the gel, or only a very faint band is detected. This occurs despite using appropriate template and primers, suggesting insufficient amplification.
PCR products appear as continuous smears rather than discrete bands on gel, indicating template degradation, excessive cycling, or nonspecific amplification throughout a size range.
Complete absence of PCR product on gel due to missing essential reaction components, representing a critical setup error.
Multiple unwanted PCR products appear on gel alongside or instead of target band, caused by reagent concentration imbalances or primer design problems.
Gel shows no amplification product or very weak bands despite correct thermal cycling parameters, indicating problems with reaction component concentrations.
PCR fails or produces weak products despite correct reagent concentrations and cycling parameters, due to compromised template DNA quality or presence of inhibitors.
After PCR amplification and gel electrophoresis, no visible band appears at the expected product size, or only a very faint band is observed, indicating insufficient or failed amplification.
No visible band or very faint band on gel despite proper thermal cycling parameters. The reaction components may be at suboptimal or inhibitory concentrations.
PCR fails to produce visible band despite correct concentrations and cycling parameters. Template degradation, contamination, or impure reagents may be inhibiting the reaction.
Multiple bands appear on gel in addition to or instead of the expected target band. Primer-dimer artifacts or nonspecific amplification products are visible, indicating lack of reaction specificity.
No amplification observed even with optimized reaction conditions. Primers may have incorrect sequences, poor design, or target sequence may be too long for current protocol.
No visible band or very faint band appears on the gel after PCR amplification. This indicates insufficient or failed amplification of the target sequence.
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