NEBNext Quick DNA Sample Prep Master Mix Set for 454 End Repair and dA-Tailing Small Fragment Removal (E6090)
Protocol
- Add the End Repaired/dA-Tailed/Adaptor ligated DNA sample directly to the previously prepared Ampure beads. Vortex briefly to mix, followed by a quick spin to collect liquid from the sides of the tube.
- Incubate at room temperature for 5 minutes.
- Place the tube on a Magnetic Separator.
- When the beads have collected to the wall of the tube and the solution is clear, remove and discard the supernatant. Be careful not to disturb the beads.
- Add 100ul of TE and vortex until the beads are completely resuspended.
- Add 500 μl of NEBNext Sizing Buffer and briefly vortex to mix.
- Incubate at room temperature for 5 minutes.
- Place the tube on a Magnetic Separator.
- When the beads are collected to the wall of the tube and the solution is clear, remove and discard the supernatant. Be careful not to disturb the beads.
- Repeat steps 5-9 one time.
- Keep the tube on the magnet and wash the beads twice with 1 ml of 70% ethanol.
- Keep the tube on the magnet, uncapped, and let the pellet air dry until there is no visible liquid on the sides of the tube. This typically takes 5 minutes.
- Remove the tube from the magnet, add 53 μl of TE, vortex to re-suspend the beads and spin briefly.
- Place the tube on the magnet, when the beads are collected to the wall of the tube, transfer 50 μl of the supernatant (library), to a new 1.7 ml micro-centrifuge tube. Be careful not to transfer any beads.
