Possibilities include:

  • 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.

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