Failure Case Library

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

All Techniques (8) 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)
PCR (Invitrogen Guide) severe

Low or No Amplification Due to Template Issues

PCR yields little to no product visible on gel electrophoresis. Expected band is absent or extremely faint despite using standard reaction conditions.

💡 6 causes ✓ 6 fixes
PCR (Invitrogen Guide) severe

Insufficient Amplification from Polymerase Issues

Weak or absent PCR product. Primers are degraded or show primer-dimer formation at the bottom of gel.

💡 4 causes ✓ 5 fixes
PCR (Invitrogen Guide) moderate

Nonspecific Amplification and Smearing on Gel

Gel shows multiple bands, smears, or high background instead of single clean product band. May include primer-dimers at bottom of gel.

💡 6 causes ✓ 6 fixes
PCR (Invitrogen Guide) moderate

Sequence Errors at PCR Product Termini

Sequencing shows errors, truncations, or unexpected sequences specifically at the 5′ or 3′ ends of amplified products.

💡 4 causes ✓ 5 fixes
PCR (Invitrogen Guide) severe

Amplification Failure from Primer Problems

No visible PCR product despite good template quality. Control reactions with different primers may work normally.

💡 4 causes ✓ 4 fixes
PCR (Invitrogen Guide) severe

Sequence Errors Within PCR Product Body

Sequencing reveals point mutations, insertions, or deletions within the amplified fragment that are not present in original template.

💡 5 causes ✓ 6 fixes
PCR (Invitrogen Guide) moderate

Amplification Failure from Suboptimal Cycling Parameters

No product or very weak bands despite proper template and primer quality. Reaction components appear functional in other assays.

💡 4 causes ✓ 4 fixes
PCR (Invitrogen Guide) critical

False Positive Amplification from Contamination

PCR products appear in negative control reactions without template. Products may appear in samples expected to be negative.

💡 4 causes ✓ 5 fixes