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

"Walks through the exact NEBNext Ultra II DNA Library Prep protocol from the failure case, showing the correct technique and expected library size increase."

New England Biolabs ★ 80

12 Quick Tips for NGS Library Preparation

"NEB's targeted tips for NGS library prep address common failure causes like input quality and inhibitors."

Illumina ★ 65

How can I tell if I sequenced through the insert? Part 1 | Illumina Video

"Explains how to interpret Bioanalyzer traces for library size, directly relevant to the symptom of unchanged fragment size."