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Amplicon Cleaning

We recommend cleaning the maximum available volume of the secondary PCR product to make sure you get optimal library recovery.

Materials

  • AMPure XP Beads for DNA Cleanup (Beckman Coulter, Brea, CA; Cat. No. A63881) — use a 1.8X ratio of beads to sample volume
  • Magnet compatible with a 96-well PCR plate
  • Filter-sterilized 70% ethanol (EtOH), freshly prepared
  • Filter-sterilized Tris/EDTA (TE) Buffer, pH 8.0 (10 mM / 0.1 mM)

Protocol

Pipet the calculated volume of beads into each well. Mix by pipetting up and down 10 times until the mixture is a uniform color. Let the plate stand at room temperature for 5 minutes.

Place the plate on the magnet for 10 minutes. Without disturbing the beads, carefully remove and discard the supernatant. If troubleshooting is anticipated, retain the supernatant in a fresh plate.

Keeping the plate on the magnet, add 200 uL of 70% EtOH without disturbing the beads. Let sit for 30 seconds (roughly the time it takes to fill a 96-well plate). Aspirate the ethanol and discard. Repeat this wash a second time.

Use a 10 uL multichannel pipette to remove residual ethanol. Leave the plate on the magnet for up to 2 minutes.

Warning

Stop drying immediately if you see cracking in the bead ring; over-dried beads are difficult to resuspend and significantly reduce yield.

Remove the plate from the magnet. Add 40 uL of TE Buffer, pipetting up and down 10 times until beads are fully resuspended. Let the plate stand at room temperature for 5 minutes.

Return the plate to the magnet for 1 minute. Carefully transfer ~35 uL of the clear supernatant (containing the purified DNA) to a new plate. It is better to leave 1–2 uL of DNA behind than to carry over any magnetic beads, as beads interfere with downstream fluorescence quantification. If troubleshooting is anticipated, retain the beads.

With cleaning complete, proceed to Quantification and Pooling.