Clean Up Libraries

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This step uses a double-sided bead purification procedure to purify the libraries.

• Illumina Purification Beads (IPB)
• Resuspension Buffer (RSB)
• Absolute ethanol (EtOH)
• Nuclease-free water
• 96-well PCR plates (2)
• IPB
– Avoid disturbing the bead pellet when washing.
– Use IPB at room temperature.
1. Prepare the following consumables:
• IPB—Vortex to mix.
• RSB—Vortex to mix.
2. Label a new 96-well PCR plate CNL1.
3. Label a new 96-well PCR plate CNL2.
4. For each reaction, combine the following volumes to prepare 200 µl fresh 80% EtOH:
• EtOH (160 µl)
• Nuclease-free water (40 µl)

These volumes produce 200 μl 80% EtOH per reaction, including overage.

Vortex to mix.

1. Remove from the thermal cycler.
2. Add 50 μl RSB to each well.
3. Add 40 μl IPB to each well.
4. Pipette to mix.
5. Incubate at room temperature for 2 minutes.
6. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
7. Transfer 127 µl supernatant from each well of the FLT plate to the corresponding well of the CNL1 plate.
8. Discard the FLT plate.
9. Add 18 μl IPB to each well of the CNL1 plate.
10. Pipette to mix.
11. Incubate at room temperature for 2 minutes.
12. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
13. Remove and discard all supernatant from each well.
14. Wash beads as follows.
a. Keep on the magnetic stand and add 180 µl fresh 80% EtOH to each well.
b. Wait 30 seconds.
c. Remove and discard all supernatant from each well.
15. Centrifuge briefly and return to the magnetic stand.
16. Using a pipette set to 20 µl, remove all residual EtOH from each well.
17. Air-dry on the magnetic stand for 2 minutes.
18. Remove from the magnetic stand and add 30 μl RSB to each well.
19. Pipette to mix.
20. Incubate at room temperature for 2 minutes.
21. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
22. Transfer 28 µl supernatant from each well of the CNL1 plate to the corresponding well of the CNL2 plate, and then place on ice.
23. Discard the CNL1 plate.

SAFE STOPPING POINT

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