Clean Up Libraries

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This step uses a 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
1. Prepare the following consumables:

Item

Storage

Instructions

IPB

15°C to 30°C

Resuspend IPB beads.

RSB

2°C to 8°C

Bring to room temperature. Vortex to mix.
2. For each sample, prepare 400 µl fresh 80% EtOH from absolute ethanol. Including an overage of 20% is recommended.
1. Use a plate shaker to shake the 96-well PCR plate at 1800 rpm for 1 minute.
2. Place the plate on the magnetic stand and wait until the liquid is clear (~1 minute).
3. Transfer 45 µl supernatant from each well of the PCR plate to the corresponding well of a new MIDI plate.
4. Resuspend Illumina Purification Beads.
5. For gDNA, blood, or saliva, perform the following steps.

For faster turnaround, use the Single IPB Methodology (Optional). The two-step, double-sided cleanup process here will produce optimal results. If speed is more important, a 1-step cleanup can be performed.

a. Add 77 µl nuclease-free water to each well-containing supernatant.
b. Add 88 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 1800 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, vortex the IPB, and then add 20 µl to each well of a new MIDI plate.
g. Remove seal and transfer 200 µl supernatant from each well of the first plate to the corresponding well of the new MIDI plate containing 20 µl IPB.
h. Pipette each well of the MIDI plate 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1800 rpm for 1 minute.
i. Discard the first plate.
6. Incubate at room temperature for 5 minutes.
7. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
8. Without disturbing the beads, remove and discard all supernatant.
9. Wash two times as follows.
a. With the plate on the magnetic stand, add 200 µl fresh 80% EtOH without mixing.
b. Wait for 30 seconds.
c. Without disturbing the beads, remove and discard supernatant.
10. Use a 20 µl pipette to remove and discard residual EtOH.
11. Air-dry on the magnetic stand for 5 minutes.
12. Remove from the magnetic stand.
13. Add 17 µl RSB to the beads.
14. Seal the plate, and then use a plate shaker at 1800 rpm for 2 minutes.
15. Incubate at room temperature for 2 minutes.
16. Place the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).
17. Transfer 15 µl supernatant to a new 96-well PCR plate.

SAFE STOPPING POINT

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