Clean Up Libraries

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This step uses double-sided bead purification procedure to purify the amplified and indexed libraries.

• IPB (Illumina Purification Beads)
• RSB (Resuspension Buffer)
• EtOH (Freshly prepared 80% ethanol)
• Nuclease-free water
• 96-well 0.8 ml Polypropylene Deepwell Storage Plate (MIDI plate) (2)
• 96-well PCR plate
• Microseal 'B' adhesive seal
• Microseal 'F' foil seal
• IPB
– Must be at room temperature before use.
– Vortex before each use.
– Vortex frequently to make sure the beads are evenly distributed.
– Aspirate and dispense slowly due to the viscosity of the solution.
3. RSB—Vortex to mix.
4. Prepare fresh 80% EtOH from absolute ethanol.
1. Centrifuge at 280 × g for 1 minute to collect contents at the bottom of the well.
2. Place the plate on the magnetic stand and wait until the liquid is clear (~5 minutes).
3. Transfer 45 µl supernatant from each well of the PCR plate to the corresponding well of a new MIDI plate.
4. 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.
5. For standard DNA input > 500 bp, perform the following steps.
a. Add 40 µl nuclease-free water to each well-containing supernatant.
b. Add 45 µl IPB to each well-containing supernatant.
c. Pipette each well 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
d. Seal the plate and incubate at room temperature for 5 minutes.
e. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
f. During incubation, thoroughly vortex the IPB (undiluted stock tube), and then add 15 µl to each well of a new MIDI plate.
g. Transfer 125 µl supernatant from each well of the first plate into the corresponding well of the new MIDI plate containing 15 µl undiluted IPB.
h. Pipette each well in the MIDI plate 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
i. Discard the first plate.
6. For small PCR amplicon input < 500 bp, perform the following steps.
a. Add 81 µl IPB to each well of the MIDI plate containing supernatant.
b. Pipette each well 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
7. Incubate the sealed MIDI plate at room temperature for 5 minutes.
8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
9. Without disturbing the beads, remove and discard supernatant.
10. Wash beads as follows.
a. With the plate on the magnetic stand, add 200 µl fresh 80% EtOH without mixing.
b. Incubate for 30 seconds.
c. Without disturbing the beads, remove and discard supernatant.
11. Wash beads a second time.
12. Use a 20 µl pipette to remove and discard residual EtOH.
13. Air-dry on the magnetic stand for 5 minutes.
14. Remove from the magnetic stand.
15. Add 32 µl RSB to the beads.
16. Pipette to resuspend.
17. Incubate at room temperature for 2 minutes.
18. Place the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).
19. Transfer 30 µl supernatant to a new 96-well PCR plate.

SAFE STOPPING POINT

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