Troubleshooting playbooks
First-party guides from the same library Diagnostics uses. Each page ends with Diagnose your run — no scraped forum copy.
PCR / Gel
- PCR / Gel: no bandsAmplification failure — no product formed in any lane.
- PCR / Gel: faint bandsLow-yield amplification — product present but under-loaded.
- PCR / Gel: primer dimersPrimers annealing to each other instead of (or in addition to) template.
- PCR / Gel: non-specific smearOff-target amplification across a broad size range.
- PCR / Gel: unexpected band sizeAmplicon runs at the wrong molecular weight versus the expected/ladder reference.
Assay / ELISA / Binding
- Assay / ELISA / Binding: high backgroundElevated signal in blank / negative-control wells.
- Assay / ELISA / Binding: low signalWeak or absent readout even in positive-expected wells.
- Assay / ELISA / Binding: saturated curveSignal plateaus across a wide concentration range instead of a sigmoidal response.
- Assay / ELISA / Binding: high cv between replicatesPoor reproducibility across technical or biological replicate wells.
- Assay / ELISA / Binding: poor standard curveStandard curve R² / shape unfit for quantification (non-linear, noisy, or inverted).
- Assay / ELISA / Binding: edge effectsPerimeter wells systematically higher or lower than interior replicates.
- Assay / ELISA / Binding: no signalNo readable OD/fluorescence above blank, including expected positive wells.
- Assay / ELISA / Binding: hook effectVery high analyte reads paradoxically low until diluted (prozone/hook).
Dry Lab / Computational
- Dry Lab / Computational: out of memory (oom)Job killed by the scheduler or OS for exceeding allocated memory.
- Dry Lab / Computational: exit code failureThe job terminated with a non-zero exit status.
- Dry Lab / Computational: low alignment / mapping rateA large fraction of reads fail to align to the reference.
- Dry Lab / Computational: unpinned dependency errorA package resolved to a version incompatible with the pipeline.
Western blot
- Western blot: no bandsNo specific immunoreactive bands on the blot, including expected positive targets.
- Western blot: high backgroundElevated membrane background obscuring specific bands.
- Western blot: uneven transferPatchy or gradient protein transfer across lanes or the membrane.
- Western blot: unexpected sizeSpecific band(s) appear at a molecular weight different from the expected size.
- Western blot: multiple bandsMore immunoreactive bands than expected for the target.
- Western blot: faint bandsSpecific bands present but weaker than expected for the load and antibody.
qPCR
- qPCR: late / no ctAmplification absent or Ct far later than expected for the input.
- qPCR: ntc amplificationNo-template control shows amplification above the lab’s Ct cutoff.
- qPCR: multiple melt peaksMelt analysis shows more than one product peak for a supposed single-amplicon assay.
- qPCR: poor efficiencyStandard-curve efficiency outside the lab’s accepted window.
- qPCR: high replicate ct cvTechnical replicate Cts disagree beyond the lab’s usual spread.
- qPCR: flat amplification curveFluorescence stays at baseline across cycles despite expected template.
Cloning
- Cloning: no coloniesTransformation plate shows no colonies (or far below expected CFU).
- Cloning: empty vectorColonies grow but screened clones lack the insert (vector only).
- Cloning: wrong insertScreened clones contain an insert of unexpected size or sequence.
- Cloning: satellite coloniesTiny satellite colonies surround larger colonies on antibiotic plates.
- Cloning: low transformation efficiencyColonies appear but CFU is far below the competent-cell rating or lab baseline.
Cell Culture
- Cell Culture: poor attachmentCells fail to attach to the culture surface within the expected window (including epithelial lines that need collagen coating).
- Cell Culture: slow growthDoubling time longer than expected for the authenticated line and media recipe.
- Cell Culture: contaminationVisual or growth-pattern signs of microbial contamination in culture.
- Cell Culture: mycoplasma suspectedSilent mycoplasma contamination suspected from chronic underperformance without visible turbidity.
- Cell Culture: cell-line misidentificationWorking culture morphology, markers, or behaviour does not match the authenticated line.
- Cell Culture: low viabilityCell viability below expected threshold on counting.
- Cell Culture: clumpingCells aggregate instead of forming a single-cell suspension or even monolayer.
Flow cytometry
- Flow cytometry: weak signalTarget fluorophore signal too low to resolve positive from negative populations.
- Flow cytometry: compensation issueSpectral spillover not correctly compensated — populations skewed or negative MFI artifacts.
- Flow cytometry: high backgroundElevated non-specific fluorescence obscuring true positives.
CRISPR
- CRISPR: low editing efficiencyIndel / knock-in rate far below the expected efficiency for this guide and delivery.
- CRISPR: off-target concernSuspected off-target edits or phenotype not explained by the on-target genotype.
- CRISPR: no clones recoveredNo single-cell clones expand after editing / selection / limiting dilution.
Protein expression
- Protein expression: no expressionNo detectable recombinant protein on gel / blot / activity after induction or transfection.
- Protein expression: insoluble proteinRecombinant protein is produced but remains in the insoluble fraction / inclusion bodies.
- Protein expression: degraded proteinProduct appears as fragments or disappears on storage / purification.
Guides: Technique hubs by category