Clean Up sgRNA Samples

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• Illumina Purification Beads 2 (IPB2)
• Absolute ethanol (EtOH)
• Nuclease-free water
• IDTE Buffer
• 0.2 ml 8-tube strips
1. Prepare the following consumables:
• IPB2—Vortex to resuspend.
2. For each reaction, prepare 400 µl fresh 85% EtOH solution from absolute EtOH.
1. Use the following table to determine the magnetic bead ratio to use based on the expected sgRNA library size.

If using a magnetic bead ratio other than 0.5–0.8X, substitute IPB2 volumes in steps 2 and 8.

Expected sgRNA Library Size (bp)

Magnetic Bead Ratio (first and second addition)

First Magnetic Bead Addition (µl)

Second Magnetic Bead Addition (µl)

> 550

0.45–0.7X

22.5

12.5

450–550

0.5–0.8X

25

15

300–450

0.6–0.9X

30

15

< 300

0.7–1.0X

35

15
2. For 50 μl reaction volume, add 25 μl IPB2. Seal with a new cap.

This is a 0.5X magnetic bead ratio. Adjust as necessary if reaction volume is not 50 µl.

3. Vortex for 10 seconds, and then centrifuge briefly (~2 seconds).
4. Incubate at room temperature for 5 minutes.
5. If any magnetic beads remain on the lid or side of the tube, centrifuge briefly.
6. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
7. Without disturbing the beads, transfer the supernatant to a new tube strip.
8. Add 15 µl IPB2.

This is a final magnetic bead ratio of 0.8X.

9. Vortex for 10 seconds, and then centrifuge briefly (~2 seconds).
10. Incubate at room temperature for 5 minutes.
11. If any magnetic beads remain on the lid or side of the tube, centrifuge briefly.
12. Place onto the magnetic stand and wait until the liquid is clear (~5 minutes).
13. Without disturbing the beads, remove and discard the supernatant.
14. Wash the beads as follows.
a. Add 200 μl fresh 85% EtOH.
b. Wait 30 seconds.
c. Without disturbing the beads, remove and discard EtOH.
15. Wash the beads a second time.
16. Without disturbing the beads, use a 20 μl pipette to remove all residual EtOH.
17. Air-dry until a slight gloss remains on the bead surface (~2 minutes).

Depending on the humidity level, air-drying can take up to 5 minutes.

Overdrying the beads can cause cracks, decrease the elution efficiency, and result in reduced cDNA yield.

18. Remove from the magnetic stand.
19. Add 22 μl IDTE Buffer.
20. Pipette 10 times at the 20 μl stroke to resuspend.
21. Incubate at room temperature for 5 minutes.

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

22. If necessary, centrifuge briefly.
23. Place tubes on the magnetic stand and wait until the liquid is clear (~2 minutes).
24. Transfer 20 μl of supernatant to a new PCR tube strip.

This contains the sequencing-ready purified sgRNA libraries.

SAFE STOPPING POINT

If stopping, store the purified sgRNA libraries at -25°C to -15°C for up to 14 days.