Bioanalyzer peaks below 85 bp detected

Symptom

Presence of Bioanalyzer peaks smaller than 85 bp observed after PCR cleanup. These peaks represent residual primers from the amplification reaction.

1. 1 Incomplete removal of primers during initial PCR cleanup step
2. 2 Insufficient bead-to-sample ratio (below 0.9X) during purification
3. 3 Inadequate washing or elution protocol during bead-based cleanup

1. 1 Perform second cleanup with 0.9X SPRIselect Beads or NEBNext Sample Purification Beads
2. 2 Note: Second cleanup may result in reduction of library yield
3. 3 Ensure adequate bead incubation time and proper washing technique
4. 4 Verify ethanol wash quality and complete removal before elution

Related Video (2)

the bumbling biochemist ★ 70

rRNA depletion strategies

"Demonstrates rRNA depletion strategy and QC context for RNA-seq library prep."

RocheSequencingUSA ★ 60

The Importance of Performing Ribosomal RNA Depletion

"Demonstrates rRNA depletion strategy and QC context for RNA-seq library prep."