Clean Up Enriched Library

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This step uses magnetic beads to purify the enriched library.

• IPB (Illumina Purification Beads)
• RSB (Resuspension Buffer)
• 96-well PCR plate, semiskirted
• Freshly prepared 80% ethanol (EtOH)
• Microseal 'B' adhesive film
• IPB
– Use at room temperature.
– Resuspend before each use.
1. Prepare the following consumables:
• RSB—Vortex and invert to mix.
• EtOH—Prepare 80% EtOH from absolute EtOH.
1. Centrifuge the sealed plate at 280 × g for 10 seconds.
2. Resuspend IPB as follows.
a. To mix, invert the bottle manually for 2 minutes, at a rate of 1 inversion per second. While inverting, rotate the bottle 90 degrees every 30 seconds.
b. If beads are still adhered to the walls of the container, invert the bottle manually for an additional 1 minute.
3. Add 90 µl IPB to each well.
4. Seal and shake at 2200 rpm for 1 minute.
5. Incubate at room temperature for 5 minutes.
6. Centrifuge at 280 × g for 10 seconds, and then unseal.
7. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
8. Remove and discard all supernatant.
9. 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.
10. Wash beads a second time.
11. With a 20 µl pipette, remove all residual EtOH.
12. Air-dry on the magnetic stand for 2 minutes.
13. Remove from the magnetic stand.
14. Add 32 µl RSB to each well.
15. Seal and shake at 2600 rpm for 1 minute.
16. Incubate at room temperature for 2 minutes.
17. Centrifuge at 280 × g for 10 seconds, and then unseal.
18. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
19. Transfer 30 µl supernatant from each well to 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 7 days.