Clean Up Amplified DNA

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This step uses Illumina Purification Beads (IPB) to purify the amplified DNA before hybridization to probes.

• Illumina Purification Beads (IPB)
• Resuspension Buffer (RSB)
• Absolute ethanol (EtOH)
• Nuclease-free water
• 1.7 ml microcentrifuge tube
• 96-well PCR plate
• 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 SSP1.
3. For each reaction, combine the following volumes to prepare 200 µl 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. Vortex IPB to resuspend.
3. Add 30 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. Remove and discard all supernatant from each well.
8. 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.
9. Centrifuge briefly and return to the magnetic stand.
10. Using a pipette set to 20 µl, remove all residual EtOH from each well.
11. Air-dry on the magnetic stand for 2 minutes.
12. Remove from the magnetic stand and add 20 μl RSB to each well.
13. Pipette to mix.
14. Incubate at room temperature for 2 minutes.
15. Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
16. Transfer 19 µl supernatant from each well of the AMP plate to the corresponding well of the SSP1 plate.
17. Quantify 2 μl DNA from each well using a Qubit dsDNA HS Assay Kit.

Expect a ≥ 3.3 ng/µl library yield.

SAFE STOPPING POINT

If you are stopping, seal the SSP1 plate with Microseal 'B' adhesive, and store at -25°C to -15°C for up to 30 days.