Failure Case Library

Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.

All Techniques (10) PCR (Sigma Guide) (12) End-point PCR Primers (9) Western Blot (9) Wound Healing Assay (5) Cell Culture (Contamination) (7) Lipid Transfection (8) CCK-8 Cell Viability Assay (5) Colony Formation Assay (10) Transfection (8) Nucleic Acid Quantification (9) Transfection (Co-transfection) (1) Protein Extraction (4) Plasmid Construction (Double Digest) (1) Sanger Sequencing (2) Plasmid Construction (1) qPCR (RT-qPCR) (5) Transwell Migration / Invasion Assay (5) Immunohistochemistry (IHC) (6) PCR (Polymerase Chain Reaction) (26) Restriction Enzyme Digest (13) DNA Cleanup & Plasmid Purification (7) RNA Cleanup (4) NGS Library Preparation (NEBNext Ultra II) (7) HMW DNA Extraction (Monarch) (7) LAMP (Loop-mediated Isothermal Amplification) (7) RNA Depletion for RNA-seq (7) Bacterial rRNA Depletion (4) Cell-free DNA Extraction (8) ELISA (Competitive) (18) ELISA (Signal Problems) (11) ELISA (High Background) (8) ELISA (Inconsistent Results / High CV) (6) ELISA (Standard Curve Fit Problems) (6) Western Blot (Weak / No Signal) (6) Western Blot (Detection Problems) (7) Western Blot (Bands at Wrong MW) (5) Western Blot (Misshapen / Uneven Bands) (5) Western Blot (Unexpected Multiple Bands) (7) Western Blot (Unusual Gel Band Appearance) (3) ChIP (High Background) (1) ChIP (Low Resolution with High Background) (6) ChIP (Low Signal) (8) ChIP (PCR Amplification Problems) (4) Immunohistochemistry (High Background) (9) Immunohistochemistry (No Staining) (9) Immunoprecipitation (High Antibody Elution) (1) Immunoprecipitation (High Background) (8) Immunoprecipitation (No Protein Detected) (5) Immunoprecipitation (Protein Obstruction) (1) ELISPOT (8) Tissue Imaging (Autofluorescence) (9) Flow Cytometry (Troubleshooting) (8) ELISA Development (9) Flow Cytometry (Autofluorescence) (7) Flow Cytometry (Compensation) (7) Flow Cytometry (Fc Blocking) (7) Flow Cytometry (Fixation & Permeabilization) (9) Flow Cytometry (Isotype Controls) (7) Flow Cytometry (Fixation Buffers) (7) Western Blot (CST Guide) (8) ChIP (CST Guide) (8) Immunoprecipitation (CST Guide) (14) Immunohistochemistry (CST Guide) (14) Flow Cytometry (CST Guide) (8) ELISA (R&D Guide) (10) ELISA (Sigma Guide) (6) Western Blot Immunodetection (19) IP-Western Blot (6) Flow Cytometry (Sample Considerations) (14) Flow Cytometry (Paraformaldehyde Fixation) (14) Western Blot (Sigma Protocol) (8) PCR / RT-PCR Amplification Problems (5) Cell Culture (Cell Death) (8) Cell Culture (Precipitates) (6) PCR (Invitrogen Guide) (8) PCR / qPCR Plastics (9) Flow Cytometry (Controls) (8) Plasmid Mini-prep (1) Transfection (siRNA Knockdown) (1) Western Blot (Blue Background) (1)
Colony Formation Assay minor

Plate scratches or impurities interfere with imaging

Imaging shows linear scratches, dust specks, or crystal violet precipitate that look like colonies or obscure them.

💡 3 causes ✓ 3 fixes
Colony Formation Assay moderate

"Halo" or edge-ring artifact around dishes

A ring of stain appears around the dish edge; cells aggregate at the periphery due to evaporation / meniscus effects.

💡 3 causes ✓ 3 fixes
Colony Formation Assay minor

Crystal violet stain is too dark — high background

Entire dish is deeply purple; individual colony boundaries are blurred by background staining.

💡 3 causes ✓ 3 fixes
Colony Formation Assay minor

Crystal violet stain is too light — colonies hard to see

Stained dish shows only very faint purple; colonies are hard to distinguish from background.

💡 3 causes ✓ 3 fixes
Colony Formation Assay moderate

Uneven colony sizes (large mixed with small)

Some colonies are very large while others are tiny — suggests cells were not properly singularized at seeding.

💡 3 causes ✓ 3 fixes
Colony Formation Assay severe

Too few colonies — sparse, hard to count statistics

Only a handful of visible colonies per dish; counts are too low to give meaningful statistics.

💡 4 causes ✓ 4 fixes
Colony Formation Assay critical

No colonies at all — total failure

After 7 – 14 days of culture, the entire dish shows no visible colonies after staining.

💡 5 causes ✓ 5 fixes
Colony Formation Assay severe

Replicate dishes vary widely — conclusion is unsupported

Three replicates of the same group give very different colony counts. Showing only a representative image without statistics is misleading.

💡 4 causes ✓ 4 fixes
Colony Formation Assay moderate

Dirty background or uneven crystal violet staining

Staining shows blotches, smears, or uneven intensity across the dish — technical artifacts can be mistaken for real biological differences.

💡 4 causes ✓ 4 fixes
Colony Formation Assay severe

Plated too dense — colonies merge into one another

Many colonies fuse together so individual colonies cannot be counted reliably; counts will be over- or under-estimated.

💡 2 causes ✓ 3 fixes