Clean Up Libraries

This step uses single-sided bead purification to purify amplified libraries.

• IPB (Illumina Purification Beads)
• EtOH (Freshly prepared 80% ethanol)
• RSB (Resuspension Buffer)
• 96-well 0.8 ml polypropylene deepwell storage plate (MIDI plate) (2)
• 96-well PCR plate
• Microseal 'B' adhesive seal
• Microseal 'F' foil seals
• Nuclease-free water

About Reagents

• Illumina Purification Beads
– Must be at room temperature before use.
– Resuspend before each use.
– Resuspend frequently to make sure that 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

-25°C to -15°C

Thaw and bring to room temperature. Vortex to mix.

RSB can be stored at 2°C to 8°C after the initial thaw.
2. Prepare fresh 80% EtOH from absolute ethanol.
1. Centrifuge at 280 × g at 20°C for 1 minute to collect contents at the bottom of the well.
2. Transfer 50 µl supernatant from each well of the PCR plate to corresponding wells of a new MIDI plate.

The ratio of supernatant to volume of IPB is 3:2. If you transfer less than 50 µl supernatant, adjust the volume of IPB accordingly.

3. If you are using standard DNA input, add 30 µl IPB to each well containing supernatant.
4. If you are using small PCR amplicon sample input, add the IPB volume according to your input size.

Input Size (bp)

IPB Recommendation

IPB Volume (µl)

300–500

1.8x IPB

90

> 500

0.6x IPB

(0.5x IPB for ≥ 2 x 250 cycles)

30

(25 µl for ≥ 2 x 250 cycles)
5. Seal the plate, and then use a plate shaker at 1800 rpm for 2 minutes.
6. Incubate at room temperature for 5 minutes.
7. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
8. Without disturbing the beads, remove and discard all supernatant.
9. Wash two times with 200 µl 80% EtOH as follows.
a. With the plate on the magnetic stand, add 200 µl fresh 80% EtOH without mixing.
b. Incubate for 30 seconds.
c. Without disturbing the beads, remove and discard all supernatant.
10. Use a 20 µl pipette to remove and discard residual EtOH.
11. Air-dry on the magnetic stand for 15 minutes.
12. Remove from the magnetic stand.
13. Add 52.5 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 on the magnetic stand and wait until the liquid is clear (~2 minutes).
17. Transfer 50 µl supernatant to a new 96-well PCR plate.

SAFE STOPPING POINT

If you are stopping, seal the plate with Microseal 'B' adhesive seal or Microseal 'F' foil seal and store at ‑25°C to ‑15°C for up to 7 days.