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 the following online FAQ for further details: Are my libraries overcycled? If so, what should I do? ).

A carryover of Purification Beads (PB) can also result in a peak around and beyond the upper marker on TapeStation/ Bioanalyzer/ Fragment Analyzer traces. 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 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.

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