- Using incompatible index primers.
- DNA quantification was inaccurate, especially if using spectrophotometric methods such as nanodrop, OR DNA quality is extremely poor.
- 30% ethanol instead of 70% ethanol OR using TE/H2O instead of 70% ethanol was used in DNA purification.
- Forgetting to add magnetic beads for any of the purification steps.
- Forgetting to add one or both index primers in the 2nd PCR step.
- Over-digestion, forgetting to add Stop Buffer after digestion, or pausing after digestion step.
- Weak or incompatible magnetic rack. Do not use racks designed for 1.5 ml tube.