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)
RNA Depletion for RNA-seq severe

No Depletion Due to Incorrect Probe Design Input

Percentage of reads mapping to the targeted sequence does not decrease after depletion treatment. Sequencing analysis shows target RNA remains at original levels.

💡 4 causes ✓ 5 fixes
RNA Depletion for RNA-seq severe

Probe Integrity Compromised During Storage or Synthesis

Target RNA depletion efficiency is significantly reduced or absent. Sequencing shows minimal reduction in targeted reads despite proper protocol execution.

💡 3 causes ✓ 4 fixes
RNA Depletion for RNA-seq severe

DNA Contamination Interferes with RNA Quantification and Depletion

RNA depletion shows poor efficiency with high residual target reads. RNA quantification may be inaccurate and inconsistent with expected values.

💡 3 causes ✓ 4 fixes
RNA Depletion for RNA-seq moderate

Chemical Contaminants Inhibit Probe Hybridization

Depletion efficiency is reduced or completely absent. Target RNA remains at high levels after treatment despite using validated probe designs.

💡 4 causes ✓ 5 fixes
RNA Depletion for RNA-seq severe

Improper Temperature Ramp During Probe Hybridization

Probe hybridization step was unsuccessful resulting in poor depletion. Target RNA removal is inconsistent or incomplete across replicate samples.

💡 4 causes ✓ 5 fixes
RNA Depletion for RNA-seq moderate

Non-Uniform Depletion Across Targeted Sequences

Depletion efficiency varies significantly across different targeted sequences. Some target regions show good depletion while others remain at high levels.

💡 4 causes ✓ 5 fixes
RNA Depletion for RNA-seq moderate

Genome Annotation Version Mismatch Between Design and Analysis

Target sequence used to design probes differs from the reference used to evaluate depletion. Depletion appears ineffective when analyzed against current genome annotations.

💡 4 causes ✓ 5 fixes