Clean Up cDNA

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• IPB2 (Illumina Purification Beads 2)
• IDTE Buffer
• Absolute ethanol (EtOH)
• Nuclease-free water (customer provided)
1. Prepare the following consumables:
• IPB2—Vortex to resuspend.
• For each reaction, prepare 6 ml fresh 85% EtOH solution from absolute EtOH.
1. For each 2800 µl reaction volume, add 2240 µl IPB2. If needed, adjust the magnetic bead volume for a 0.8X ratio of IPB2.
2. Vortex until thoroughly mixed and homogenous, then pulse centrifuge to bring the liquid to the bottom of the tube.
3. Incubate at room temperature for 5 minutes.
4. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).

If magnetic beads do not pellet after 5 minutes, there might be PIP carryover. Leave on the magnetic stand until liquid is clear.

5. Without disturbing the beads, remove and discard supernatant.
6. Wash beads as follows.
a. Add 3 ml fresh 85% EtOH.
b. Wait 30 seconds.
c. Without disturbing the pellet, remove and discard 3 ml EtOH.
7. Wash the beads a second time.
8. Without disturbing the beads, use a 20 µl pipette to remove residual EtOH.
9. Air-dry until a slight gloss remains on the bead surface (~5 minutes).

Depending on humidity level, air-drying can take up to 10 minutes.

Overdrying the beads can result in cracks and decrease elution efficiency.

10. Remove from the magnetic stand.
11. Add 162 μl IDTE Buffer.
12. Pipette to resuspend.
13. Pulse centrifuge to bring the liquid to the bottom of the tube.
14. Incubate at room temperature for 5 minutes.

If bead pellets are overdried, pipette mix frequently during incubation to minimize sample loss.

15. Place into the magnetic stand and wait until the mixture is clear (~2 minutes). Keep the 5 ml tubes on the magnetic stand.
16. Prepare 4 aliquots per sample of 40 μl supernatant into separate PCR tubes.

This cDNA is used as input for library preparation. The remaining 2 µl supernatant on the magnetic stand is used for QC.

17. Store the cDNA for library preparation at -85°C to -65°C for up to 72 hours.

Storing cDNA in a high amplicon-contaminated area might affect the quality of your samples. Storing cDNA for longer than 72 hours can result in final library QC failure from degraded cDNA.