This video offers tips for improving your NGS library prep bead cleanups with pipetting recommendations.
Script
During a bead cleanup, magnetic beads are used to bind nucleic acids. If you accidentally remove some of the beads in the course of a bead clean-up step, you lose some of your sample, impacting your final yield. Supernatant removal is a balance between completely removing your unwanted liquid phase and maintaining the integrity of your bead pellet. For the best results, use the micropipettes that you are most comfortable with. Load a 200 μl tip on your micropipette. Next bring the magnetic rack and tubes to near eye-level so you have a good vantage point from which to precisely pipet. Prevent shifting of the tubes in the magnet while pipetting. Before introducing your pipette tip to the sample, depress the plunger past the first stop. Then, insert the pipette tip just below the surface the supernatant. Avoid disturbing the bead pellet. Slowly raise the plunger on the pipette, moving the pipette tip down as the volume level decreases. Pushing past the first stop on the pipette ensures you have excess suction to remove all liquid from the tube. Once supernatant has been completely aspirated, before you eject the tip containing supernatant, hold the filled tips against a white sheet of paper. Residual beads will be easily identifiable as a small brown spot inside of the tips. If residual beads are observed, redispense the supernatant back into the same tube. Remove the tubes from the magnetic rack, perform a quick spin to displace any bubbles at the bottom, and place sample tubes back on the rack. Using a new 200 μl tip, carefully remove the supernatant again. Check the tip again to make sure no beads are present and discard the tip. Using a 10 μl pipette, remove any excess supernatant or ethanol wash from the bottom of the tube. Often after the initial volume of wash is removed, 1-2 μl may collect on the bottom of the tube. In most SPRI-bead cleanup steps you will first remove the sample supernatant after the binding step and then again after two ethanol washes. If not all of the sample supernatant has been removed, this can lead to adaptor dimer or leftover primers in your final libraries. Residual ethanol can inhibit effective elution of your libraries from the beads. One last tip: Do not let the beads dry out…remove liquid only from as many samples as you can comfortably do before the beads dry out. Thanks for watching. If you have any other questions reach out to us at info@neb.com
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