What does this peak above the upper marker of my library trace mean?

Elevated baseline above the upper marker on TapeStation traces can indicate overcycling (see Are my libraries overcycled? What should I do? for further details).

A carryover of Purification Beads (PB) results in can also produce a peak around and beyond the upper marker of the TapeStation and also on Bioanalyzer. This can be avoided by taking care not to transfer any beads after the final elution.

A few tips to prevent bead carry-over:

• Leave approximately 2 µl of the eluate on the beads when removing the eluate for QC and sequencing. i.e., Transfer only 15 – 17 μl of the eluate into a fresh PCR plate or tube. and do not try to transfer the complete sample, as this will lead to bead carryover.

• Put your samples once again on the magnet and incubate for 5 minutes.

• Transfer 15-17 μl of the supernatant into a fresh PCR plate.

• Do not try to transfer the complete sample, as this will lead to bead carryover.

• Put your samples once again on the magnet when taking an aliquot to run for QC on Tapestation/Bioanalyzer/Fragment Analyzer, or similar.