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