Single IPB Methodology (Optional)

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The Single IPB Methodology uses the bead purification procedure to purify the amplified pre-enrichment libraries. It is an alternative to the standard double-sided IPB Clean Up Libraries.

• Single IPB cleanup removes the need of pre-enrichment library concentration before the hybridization step, which could save about 20 minutes.
• This methodology gives better diversity and lowers the number of duplicate fragments. However, it may associate with a larger deviation of percent on-target reads due to a wider range of fragment sizes.
• Double-sided IPB cleanup and size selection provides more uniform fragment size and on target metrics.
• IPB (Illumina Purification Beads)
• RSB (Resuspension Buffer)
• Freshly prepared 80% ethanol (80% EtOH)
• Nuclease-free water
• 96-well 0.8 ml Polypropylene deep-well storage plate (MIDI plate) (2)
• 96-well PCR plate
• 1.7 ml microcentrifuge tubes
• Microseal 'B' adhesive seal
• Microseal 'F' foil seal
• IPB
– Must be at room temperature before use.
– Vortex before each use.
– Vortex 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

Let stand at room temperature for 30 minutes. Vortex and invert to mix.

RSB

2°C to 8°C

Thaw and bring to room temperature. Vortex to mix.
2. For each sample, prepare 400 μl fresh 80% EtOH from absolute ethanol. Including an overage of 20% is recommended.
3. If purifying your gDNA sample before the library preparation, start at step 4 and then add 1.8x IPB to your gDNA sample. For example, if your gDNA sample volume is 50 µl, add 90 µl IPB. Then proceed to step 6 (mix IPB and samples) and continue with the rest of the procedure.
1. Shake the 96-well PCR plate at 1800 rpm for 1 minute.
2. Place the plate on the magnetic stand and wait until the liquid is clear (~1 minute).
3. Transfer 45 µl supernatant from each well of the PCR plate to the corresponding well of a new MIDI plate.
4. Vortex and invert IPB multiple times to resuspend.
5. Add 81 µl IPB to each MIDI plate well containing supernatant.
6. Pipette each well 10 times to mix. Alternatively, seal the plate and shake at 1800 rpm for 1 minute.
7. Incubate the sealed MIDI plate at room temperature for 5 minutes.
8. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
9. Without disturbing the beads, remove and discard all supernatant.
10. Wash two times 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 supernatant.
11. Use a 20 µl pipette to remove and discard residual EtOH.
12. Air-dry on the magnetic stand for 5 minutes.
13. Remove from the magnetic stand.
14. Add 17 µl RSB to the beads.
15. Seal the plate, and then use a plate shaker to shake at 1800 rpm for 2 minutes.
16. Incubate at room temperature for 2 minutes.
17. Place the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).
18. Transfer 15 µl supernatant to a new 96-well PCR plate.
19. Seal the plate.

SAFE STOPPING POINT

If you are stopping, store at -25°C to -15°C for up to 30 days.