Capture Hybridized Probes

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This step uses SMB3 (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted regions of interest. Heated washes remove nonspecific DNA binding from the beads. The enriched library is then eluted from the beads and prepared for amplification.

• EEW (Enhanced Enrichment Wash)
• EPM4 (Enhanced PCR Mix)
• RSB (Resuspension Buffer)
• SMB3 (Streptavidin Magnetic Beads)
• 1.7 ml microcentrifuge tube
• 96-well PCR plate, semiskirted
• Microseal 'B' adhesive seals

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Ventilation should be appropriate for handling of hazardous materials in reagents. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

• EEW must be at room temperature before use.
• SMB3:
– Make sure to use SMB3 and not Illumina Purification Beads for this procedure.
– SMB3 must be at room temperature before use.
– SMB3 might form a bead pellet during centrifuge steps. If pelleting occurs, pipette-mix to fully resuspend the beads, and then repeat the centrifuge step.
1. Prepare the following consumables:
• SMB3:
a. Vortex to resuspend.
b. If the bead pellet is still observed, pipette up and down to release the pellet, and then repeat the vortex.
• EEW—Vortex to mix. 

If precipitates are observed, vortex until dissolved.

• RSB—Vortex briefly.
• EPM4—Vortex to mix, and then centrifuge briefly.
2. Combine the following volumes to prepare the PCR Elution Mix. Multiply each volume by the number of samples.
• RSB (27.5 μl)
• EPM4 (22 μl)

These volumes produce 49.5 μl PCR Elution Mix per sample, which includes extra volume for accurate pipetting.

3. Vortex PCR Elution Mix to mix, and then place on ice.
4. Label a new 96-well semiskirted PCR plate ELU.
5. Save the following INC58 program on the thermal cycler:
• Choose the preheat lid option and set to 70°C
• Set the reaction volume to 100 μl
• Hold at 58°C

Bind

1. Remove the HYB plate from the thermal cycler and centrifuge briefly.
2. Vortex SMB3 for 1 minute.
3. Add 62 µl SMB3 to each well of the HYB plate.
4. Seal and shake at 1800 rpm for 4 minutes.
5. If splashing occurs, centrifuge for 5 seconds. Make sure that the beads do not pellet.
6. Place on the preprogrammed thermal cycler and run the INC58 program. Incubate at 58°C for 15 minutes.

Do not terminate the thermal cycler program until after the final incubation of the elute step.

7. If splashing occurs, centrifuge briefly.
8. Place on a peg magnetic stand and wait until the liquid is clear (2 minutes).
9. Without disturbing the beads, use a pipette set to 100 μl to remove and discard all supernatant from each well.

Wash

1. Vortex EEW for 1 minute.
2. Wash the beads as follows.
a. Remove the HYB plate from the magnetic stand.
b. Add 75 μl EEW to each well.
c. Seal and shake at 1800 rpm for 4 minutes.
d. Centrifuge briefly.
e. Place on the preprogrammed thermal cycler and run the INC58 program. Incubate at 58°C for 10 minutes.
f. Remove from the thermal cycler and centrifuge briefly.
g. Place on a peg magnetic stand and wait until the liquid is clear (2 minutes).
h. Without disturbing the beads, use a pipette to remove and discard all supernatant from each well.
3. Repeat step 2 to wash the beads a second time.
4. Use a 20 μl pipette to remove residual supernatant from each well.

Elute

1. Remove the HYB plate from the magnetic stand.
2. Add 100 μl EEW to each well.
3. Seal and shake at 1800 rpm for 4 minutes.
4. If splashing occurs, centrifuge briefly.
5. Transfer the entire volume of the resuspended bead solution to the ELU plate.

Transferring samples to the new PCR plate is a critical step that minimizes the carryover of residual reagents that can inhibit downstream PCR.

6. Discard the HYB plate.
7. Seal the ELU plate.
8. Place on the preprogrammed thermal cycler and run the INC58 program. Incubate at 58°C for 5 minutes.
9. Remove from the thermal cycler and centrifuge briefly.
10. Place on a peg magnetic stand and wait until the liquid is clear (2 minutes).
11. Without disturbing the beads, use a pipette to remove and discard all supernatant from each well.
12. Seal and centrifuge briefly.
13. Place on the peg magnetic stand for 30 seconds.
14. Use a 20 μl pipette to remove residual supernatant from each well.
15. Vortex PCR Elution Mix briefly to mix.
16. Remove the ELU plate from the magnetic stand.
17. Add 45 μl PCR Elution Mix to each well.
18. Seal and shake at 1800 rpm for 1 minute.

Visually inspect each well. If a bead pellet is still observed, pipette up and down to release the pellet.

19. Centrifuge briefly.