Residual Adaptor or Primers After PCR (NGS Library Preparation (NEBNext Ultra II))

Question

Accepted Answer

Adaptor or primer peaks visible on Bioanalyzer or similar instrument after PCR amplification, indicating inefficient cleanup or excess reagent usage. Common causes: 1) Excess adaptor used during ligation step relative to sample input 2) Excess PCR primer added or inefficient post-PCR cleanup 3) SPRI bead cleanup ratio insufficient to remove small adaptor/primer fragments Solutions: 1) Perform another 0.9X SPRI bead cleanup to remove residual adaptors and primers 2) Optimize adaptor concentration during ligation based on input amount 3) Verify correct PCR primer volume is being added according to manual specifications