Failure Case Library

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

All Techniques (7) 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)
Flow Cytometry (Compensation) severe

Antibody fails to bind compensation beads

Compensation beads show no or minimal fluorescent signal when stained with antibody, preventing creation of valid single-stain controls for compensation matrix calculation.

💡 4 causes ✓ 4 fixes
Flow Cytometry (Compensation) severe

Compensation beads saturate detectors causing overflow

Compensation bead populations appear off-scale or saturate detectors, producing signals outside the linear detection range and preventing accurate compensation matrix calculation.

💡 4 causes ✓ 4 fixes
Flow Cytometry (Compensation) severe

Tandem dye degradation on compensation beads

Tandem fluorophore compensation beads show spectral shift or altered emission profile over time, causing incorrect spillover calculation and poor compensation in acceptor channels.

💡 4 causes ✓ 5 fixes
Flow Cytometry (Compensation) critical

Incorrect compensation beads for fixable viability dyes

Fixable viability dyes (LIVE/DEAD, Zombie dyes) show no signal on standard antibody-capture beads or produce inconsistent compensation when using stained cells, causing spillover errors into viability channels.

💡 4 causes ✓ 5 fixes
Flow Cytometry (Compensation) severe

Compensation bead staining conditions mismatch experimental protocol

Compensation matrix fails to correctly remove spillover from biological samples despite proper bead staining, resulting in false-positive populations or residual spillover in multicolor panels.

💡 5 causes ✓ 5 fixes
Flow Cytometry (Compensation) severe

Poor spectral reference controls for unmixing algorithms

Spectral unmixing (Cytek Aurora, Sony ID7000) produces residual spillover or negative populations despite using compensation beads, indicating unreliable full-spectrum reference signatures.

💡 5 causes ✓ 5 fixes
Flow Cytometry (Compensation) moderate

Dim or inconsistent compensation bead staining

Compensation beads display weak fluorescence or show highly variable signal intensity between replicates, producing unreliable compensation controls and inconsistent spillover correction.

💡 5 causes ✓ 5 fixes