Isolate SNT cDNA from PIPs

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• cDNA Extraction Buffer (CEB)
• Illumina Purification Beads 2 (IPB2)
• Absolute ethanol (EtOH)
• Nuclease-free water
• IDTE Buffer
• 5 ml PCR Clean centrifuge tubes
• 0.2 ml 8-tube strips
1. Prepare the following consumables:
• IPB2—Vortex to resuspend.
2. For each reaction, prepare 6 ml fresh 85% EtOH solution from absolute EtOH.
3. Label an 8-tube strip SNT cDNA.

Isolate SNT cDNA

1. Briefly centrifuge the tubes.
2. Add 110 µl CEB to each SNT amplification reaction in the 8-tube strips.
3. Transfer the 24 aliquots of each sample into a new 5 ml tube per sample.
4. Centrifuge the 8-tube strips briefly, and then transfer any remaining beads and supernatant to the appropriate 5 ml sample tube.
5. Vortex each 5 ml tube to mix, and then centrifuge in a swinging bucket rotor at 750 × g for 2 minutes (~70% maximum braking speed).
6. Without disturbing the PIP pellet, transfer 2000 μl supernatant from each reaction into a new 5 ml tube per sample.

To remove 2000 μl supernatant, transfer 500 μl four times.

PIP carryover in the supernatant phase can adversely affect the magnetic bead cleanup.

7. Briefly centrifuge the tube containing the supernatant, and then examine the bottom of the tubes for PIPs.
8. If residual PIPs are present, transfer the supernatant to a new 5 ml tube, and then measure the transferred supernatant volume.

If the final volume of supernatant is not 2000 µl per sample, adjust the magnetic bead volume during magnetic bead purification.

Magnetic Bead Purification-Double Sided

1. For each 2000 μl reaction volume, add 1200 μl IPB2. If needed, adjust the magnetic bead volume for a 0.6X ratio of magnetic beads.
2. Vortex for 10 seconds, and then centrifuge briefly (~2 seconds).
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, transfer supernatant to a new 5 ml tube.
6. Add 800 μl IPB2 to the new tube with the supernatant.
7. Vortex for 10 seconds, and then centrifuge briefly (~2 seconds).
8. Incubate at room temperature for 5 minutes.
9. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
10. Without disturbing the beads, remove and discard supernatant.
11. Wash the beads as follows.
a. Add 3 ml fresh 85% EtOH.
b. Wait 30 seconds.
c. Without disturbing the beads, remove and discard 3 ml EtOH.
12. Wash beads a second time.
13. Without disturbing the beads, use a 20 µl pipette to remove all residual EtOH.
14. Air-dry until a slight gloss remains on the bead surface (~5 minutes).

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

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

15. Remove from the magnetic stand.
16. Add 102 μl IDTE Buffer.
17. Pipette 10 times at the 100 μl stroke to resuspend.
18. Centrifuge briefly in a swinging bucket rotor at 750 × g (~2 seconds).
19. Incubate at room temperature for 5 minutes.

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

20. Place tubes on the magnetic stand and wait until the mixture is clear (~2 minutes).
21. Transfer 100 μl supernatant to the SNT cDNA tube strip. Place on ice until you are ready to index SNT samples.

SAFE STOPPING POINT

If you are stopping, store purified SNT cDNA at -25°C to -15°C for up to 14 days.