Complete Library Preparation Failure

Symptom

No library visible on Bioanalyzer or similar instrument after amplification, or library fragments remain the same size as input DNA instead of showing expected ~120 bp increase in size.

1. 1 Input DNA contains PCR inhibitors or contaminants
2. 2 Critical reagent omitted during one or more enzymatic steps
3. 3 Reagents have become inactive due to improper storage temperature
4. 4 Complete failure of any enzymatic step in the workflow

1. 1 Ensure DNA does not contain inhibitors; consider additional cleanup step before library prep
2. 2 Confirm all reagents were added for each step in the protocol using a checklist
3. 3 Verify reagents have been stored at the appropriate temperature (typically -20°C)
4. 4 Repeat library preparation with fresh reagents and verified DNA quality

Related Video (3)

New England Biolabs ★ 95

NEBNext Ultra II DNA Library Prep Protocol

"Directly demonstrates the NEBNext Ultra II protocol, which is the exact technique failing."

New England Biolabs ★ 80

12 Quick Tips for NGS Library Preparation

"Provides practical tips for NGS library prep that can help troubleshoot or avoid contamination-related failures."

YouTube (Curated Tutorials) ★ 70

2) Next Generation Sequencing (NGS) - Sample Preparation

"Explains general NGS sample preparation steps, offering context for where inhibitors or contaminants disrupt the workflow."