Clean Up DNA

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This step uses Illumina Purification Beads to purify the amplified DNA.

• Illumina Purification Beads (IPB)
• Resuspension Buffer (RSB)
• Absolute ethanol (EtOH)
• Nuclease-free water
• 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. For each reaction, combine the following volumes to prepare 80% EtOH.
• EtOH (400 µl)
• Nuclease-free water (100 µl)

These volumes produce 500 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 100 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).