Sample Loss During SPRI Bead Cleanup (NGS Library Preparation (NEBNext Ultra II))

Question

Accepted Answer

Significantly lower library yield after SPRI bead cleanup steps, with visible reduction in final library concentration disproportionate to expected recovery rate. Common causes: 1) SPRI beads dried out completely before elution (beads turn lighter brown and start cracking) 2) Incomplete ethanol removal during SPRI bead wash leaving residual ethanol 3) Sample loss during mixing: droplets clinging to pipette tip interior not recombining with sample 4) Accidental removal of beads with supernatant during aspiration steps 5) Insufficient mixing with only partial volume or formation of bubbles reducing mixing efficiency Solutions: 1) Add Elution Buffer and mix before beads turn lighter brown and start cracking; do not over-dry beads 2) After last ethanol wash, quickly spin tube, keep on magnet and remove residual ethanol with p10 tip 3) Mix slowly to avoid droplets on tip interior; dispense last mix slowly, wait 1 second before pushing to second stop 4) When removing supernatant, check tip over white paper for beads; if visible, dispense back and allow beads to resettle 5) Mix samples well with 80-90% of total volume by pipetting up and down (10 cycles for enzymatic steps); keep tip in liquid to avoid bubbles