Purify the amplified Library:
Perform a two-round purification process with the Agencourt™ AMPure™XP Reagent:
•First round at 0.5X bead-to-sample-volume ratio: High molecular-weight DNA is bound to beads, while amplicons and primers remain in solution. Save the supernatant.
•Second round at 1.2X bead-to-original-sample-volume ratio: Amplicons are bound to beads, and primers remain in solution. Save the bead pellet, and elute the amplicons from the beads.
First-round purification:
1. Tap the plate gently on a hard flat surface or centrifuge briefly to collect the contents at the bottom of the wells, then remove the plate seal.
2. Add 25 µL (0.5X sample volume) of Agencourt™ AMPure™XP Reagent to each plate well containing ~50 µL of sample. Mix the bead suspension with the DNA thoroughly by pipetting up and down 5times.
3. Incubate the mixture for 5 minutes at room temperature.
4. Place the plate in a magnet such as the DynaMag™–96 Side Magnet for at least 5 minutes, or until the solution is clear.
5. Carefully transfer the supernatant from each well to a new well of the 96-well PCR plate without disturbing the pellet.
Second-round purification:
1. To the supernatant from step 4 above, add 60 µL (1.2Xoriginal sample volume) of Agencourt™ AMPure™ XP Reagent. Pipet up and down 5 times to mix the bead suspension with the DNA thoroughly.
2. Incubate the mixture for 5 minutes at room temperature.
3. Place the plate in the magnet for 3 minutes or until the solution is clear. Carefully remove, then discard the supernatant without disturbing the pellet.
IMPORTANT! The amplicons are bound to the beads. Save the bead pellet.
4. Add 150 µL of freshly prepared 70% ethanol to each well, then move the plate side to side in the magnet to wash the beads. Remove and discard the supernatant without disturbing the pellet.
6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in the magnet, air-dry the beads at room temperature for 2−5 minutes. Do not over dry.
7. Remove the plate from the magnet, then add 50 µL of Low TE to the pellet to disperse the beads.
8. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, then centrifuge to collect droplets.
9. Incubate at room temperature for at least 2 minutes.
10. Place the plate in the magnet for at least 2 minutes, then analyze an aliquot of the supernatant.