Clean Up Library

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This step uses magnetic beads to purify the dual-indexed libraries.

• RSB (Resuspension Buffer)
• Agencourt AMPure XP
• Freshly prepared 80% EtOH
• 96-well PCR plate, semiskirted
• Microseal 'B' adhesive film
1. Prepare the following consumables:

Reagent

Storage

Instructions

AMPure XP

2°C to 8°C

Let stand for 30 minutes to bring to room temperature. Vortex and invert to mix.

RSB

-25°C to -15°C

Thaw at room temperature. Vortex and invert to mix.
1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.

2. Vortex AMPure XP to resuspend.

3. Add 50 µl AMPure XP to each well.

4. Mix using either method:
• Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.
• Slowly pipette until the beads are resuspended.

5. Incubate at room temperature for 5 minutes.
6. Place on the magnetic stand and wait 5 minutes.
7. Remove and discard 90 µl supernatant.
8. Wash beads as follows.
a. Keep on the magnetic stand and add 175 µl fresh 80% EtOH to each well.
b. Wait 30 seconds.
c. Remove and discard all supernatant.
9. Wash beads a second time.
10. With a 20 µl pipette, remove all residual EtOH.
11. Air-dry on the magnetic stand for 2 minutes. Do not overdry the beads.
12. Remove from the magnetic stand.
13. Add 17 µl RSB to each well.

14. Mix using either method:
• Seal and shake at 2200 rpm for 1 minute.
• Slowly pipette until the beads are resuspended, and then seal.

15. If shaking did not fully resuspend the beads, slowly pipette until the beads are resuspended, and then seal.
16. Incubate at room temperature for 2 minutes.
17. Centrifuge at 280 × g for 10 seconds.
18. Place on the magnetic stand and wait 2 minutes.
19. Transfer 15 µl supernatant from each well to the corresponding well of a new PCR plate.

Safe Stopping Point

If you are stopping, seal the plate and store at -25°C to -15°C for up to 30 days.