Complete Library Preparation Failure (NGS Library Preparation (NEBNext Ultra II))

Question

Accepted Answer

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. Common causes: 1) Input DNA contains PCR inhibitors or contaminants 2) Critical reagent omitted during one or more enzymatic steps 3) Reagents have become inactive due to improper storage temperature 4) Complete failure of any enzymatic step in the workflow Solutions: 1) Ensure DNA does not contain inhibitors; consider additional cleanup step before library prep 2) Confirm all reagents were added for each step in the protocol using a checklist 3) Verify reagents have been stored at the appropriate temperature (typically -20°C) 4) Repeat library preparation with fresh reagents and verified DNA quality