Clean Up Amplified Enriched Library

This snippet causes dropdowns to open on page load. This text/paragraph does not appear in the output.

This step uses IPB (Illumina Purification Beads) to purify the enriched libraries.

• IPB (Illumina Purification Beads)
• RSB (Resuspension Buffer)
• Absolute ethanol (EtOH)
• Nuclease-free water
• 96-well PCR plates, LoBind (2)
• Microseal 'B' adhesive seals
• IPB:
– Use IPB at room temperature.
– Aspirate and dispense IPB slowly due to the viscosity of the solution.
1. Prepare IPB—Vortex for one minute to disperse the beads.
2. Combine the following volumes to prepare 80% EtOH. Multiply each volume by the number of samples being processed.
• EtOH (400 μl)
• Nuclease-free water (100 μl)

These volumes produce 500 μl 80% EtOH per sample, which includes extra volume for accurate pipetting.

3. Vortex 80% EtOH to mix.
4. Label a new 96-well LoBind PCR plate Clean Up.
5. Label a new 96-well LoBind PCR plate PL.

Bind

1. Vortex IPB briefly to resuspend the beads.
2. Add 40.5 µl IPB to each well of the Clean Up plate.
3. Remove the ELU plate from the thermal cycler and centrifuge briefly.
4. Place the ELU plate on a bar magnetic stand for 1 minute.
5. Transfer 45 μl supernatant from each well of the ELU plate to the corresponding well of the Clean Up plate, and pipette 10 times to mix.
6. Discard the ELU plate.
7. Incubate the Clean Up plate at room temperature for 5 minutes.

Wash

1. Place the Clean Up plate on a bar magnetic stand for 2 minutes.
2. Without disturbing the beads, remove and discard all supernatant from each well.
3. Wash the beads as follows.
a. Keep on the magnetic stand and add 150 µl 80% EtOH to each well.
b. Wait 30 seconds.
c. Without disturbing the beads, remove and discard all supernatant from each well.
4. Repeat step 3 to wash the beads a second time.
5. Use a 20 μl pipette to remove residual supernatant from each well.

Elute

1. Remove the plate from the magnetic stand.
2. Add 35 µl RSB to each well.
3. Seal and shake at 2200 rpm for 1 minute.

Visually inspect each well. If a bead pellet is still observed, pipette up and down to release the pellet.

4. Centrifuge briefly.
5. Incubate at room temperature for 2 minutes.
6. Place on a bar magnetic stand and wait until the liquid is clear (2 minutes).
7. Transfer 30 µl supernatant from each well of the Clean Up plate to the corresponding well of the PL plate.
8. Discard the Clean Up plate.

Safe stopping point

If you are stopping, apply Microseal ‘B’ to the PL plate, and briefly centrifuge at 280 × g. Store at 2°C to 8°C overnight, or at -25°C to -15°C for up to 30 days.