FAQ: How do I clean up the reaction once complete?

The module is designed to go directly into the NEBNext Ultra II Ligation Module (#E7595), therefore there is no cleanup step required. For use with non-Ultra II workflows, use the following protocol for cleanup using SpriSelect or AMPure XP® Beads (Beckman Coulter, Inc.):
  1. For use with AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes prior to use.
  2. Vortex beads to resuspend.
  3. Add 108 μl (1.8X) of resuspended beads to the ligation reaction. Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times.
  4. Incubate for 5 minutes at room temperature.
  5. Put the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.
  6. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
  7. Repeat Step 6 once.
  8. Air dry beads for 5 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  9. Remove the tube/plate from the magnet. Elute the DNA target from the beads by adding 47 μl of 10 mM Tris-HCl or 0.1X TE.
  10. Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature.
  11. Put the tube/PCR plate in the magnetic stand until the solution is clear.Without disturbing the bead pellet, carefully transfer 42 μl of the supernatant to a fresh, sterile microfuge tube