Capture Hybridized Probes

This step uses Streptavidin Magnetic Beads (SMB3) to capture probes hybridized to the target regions of interest within the libraries. Heated washes remove nonspecific binding from the beads. The enriched library is then eluted from the beads.

• EE1 (Enrichment Elution Buffer 1)
• EEW (Enhanced Enrichment Wash)
• ET2 (Elute Target Buffer 2)
• HP3 (2N NaOH)
• SMB3 (Streptavidin Magnetic Beads)
• One of the following containers:
– [Plate] 96-well MIDI plate and 96-well PCR plate
– [Tube] 8-tube strip
• One of the following seals:
– [Plate] Microseal 'B' adhesive seal
– [Tube] 8-tube strip caps
• One of the following magnetic stands:
– [Plate] Magnetic Stand-96
– [Tube] Magnetic Stand (8-tube strip)
• EEW
– Can be cloudy after reaching room temperature
– Can appear yellow
– Heat before use as instructed
• SMB3
– Make sure to use SMB3 and not Illumina Purification Beads for this procedure
– SMB3 must be at room temperature before use
1. Preheat the microheating system to 58°C, and then prepare the following consumables.

Item

Storage

Instructions

SMB3

2°C to 8°C

Let stand for 30 minutes to bring to room temperature. Vortex to mix before use.

EEW (amber tube)

‑25°C to ‑15°C

Thaw at room temperature. Vortex three times for 30 seconds each.

The reagent is heated during the procedure.

EE1

‑25°C to ‑15°C

Thaw at room temperature. Pipette to mix. Centrifuge briefly before use.

HP3

‑25°C to ‑15°C

Thaw at room temperature. Vortex to mix. Centrifuge briefly before use.

ET2

2°C to 8°C

Bring to room temperature. Vortex to mix. Centrifuge briefly before use.
2. Preheat a minimum of one microheating system with a heat block insert to incubate the sample plate to 58°C. An optional second microheating system can be used to preheat EEW.
3. Place the EEW (amber tube) on its side, on the heat block.

The tube remains on the heat block until the Wash procedures.

4. If the thermal cycler has an incubator option, set it to 58°C. If it does not have an incubator option, save the following program:
a. Choose the preheat lid option and set to 70°C.
b. Reaction volume is 100 μl.
c. Hold at 58°C.

Capture

1. Centrifuge the sample plate or tube at 280 × g for 10 seconds.
2. Vortex SMB3 to resuspend.
3. Add 62.5 µl SMB3 to each well or tube, and then mix thoroughly as follows.
4. Slowly pipette until the beads are resuspended, and then seal.
5. Place the sample plate or tube in the 58°C thermal cycler, close the lid, and incubate for 15 minutes.

The thermal cycler runs continuously through the capture and four washes.

Proceed to step 6 while the sample incubates.

6. Immediately centrifuge the sample plate or tube at 280 × g for 10 seconds.
7. Immediately place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8. Remove and discard all supernatant from each well or tube.

Wash

1. Remove from the magnetic stand.
2. Add 50 μl preheated EEW (amber tube) to each well or tube. Mix thoroughly as follows.
• If using a tube, cap the tube and then vortex at high speed 3 times for 10 seconds each. Do not centrifuge.
• If using a plate, seal and shake at 2400 rpm for 4 minutes.
3. Return unused EEW to the microheating system and keep heated.
4. Place the sample plate or tube on the thermal cycler and incubate for 5 minutes at 58°C.
5. Tube] Centrifuge for 3 seconds.
Plate] Centrifuge at 280 x g for 3 seconds.
6. Immediately place the plate or microcentrifuge tube on a magnetic stand and wait until the liquid is clear (~2 minutes).
7. Remove and discard all supernatant from each well or tube.
8. Repeat steps 1–7 two additional times for a total of 3 washes.

Transfer Wash

1. Remove the plate or tube from the magnetic stand.
2. Add 50 μl preheated EEW (amber tube) to each well or tube. Mix thoroughly as follows.
• If using a tube, cap the tube, and then vortex at high speed 3 times for 10 seconds each. Do not centrifuge.
• If using a plate, seal and shake at 2400 rpm for 4 minutes.
3. Transfer 50 µl to a new MIDI plate or new tube strip.

Transferring the reagent minimizes carryover of residual reagents that can inhibit downstream PCR.

4. Place the sample plate or tube on the thermal cycler and incubate for 5 minutes at 58°C.
5. [Tube] Centrifuge for 3 seconds.
Plate] Centrifuge at 280 x g for 3 seconds.
6. Immediately place on a magnetic stand and wait until the liquid is clear (~2 minutes).
7. Remove and discard all supernatant from each well or tube.
8. Centrifuge the plate or the tube at 280 × g for 30 seconds.
9. Place on a magnetic stand for 10 seconds.
10. Use a 20 µl pipette to remove and discard residual liquid from each well or from the tube.
11. Immediately proceed to Elute to prevent excessive drying of the beads and library yield loss.

Elute

1. Combine the following volumes to prepare an Elution Master Mix. Multiply each volume by the number of samples being processed.

Additional reagent is included in the volume to ensure accurate pipetting due to the potential of reagent foaming.

• EE1 (28.5 µl)
• HP3 (1.5 µl)
2. Pipette the Elution Master Mix thoroughly to mix, and then set aside at room temperature.
3. Remove from the magnetic stand.
4. Add 23 µl Elution Master Mix to each well or to the tube, and then mix thoroughly as follows.
• [Plate] Seal plate and shake at 1800 rpm for 2 minutes.
• [Tube] Cap the tube, and then vortex at high speed 3 times for 10 seconds each.
5. Incubate the plate or tube at room temperature for 2 minutes.
6. Centrifuge at 280 × g for 30 seconds.
7. Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8. Transfer 21 µl supernatant from the MIDI plate or tube strip to the corresponding well of a new 96-well PCR plate or a new 8-tube strip.
9. Add 4 μl ET2 to each well or to the tube containing 21 μl supernatant.
10. Set pipette to 20 µl and slowly pipette each well or the tube 10 times to mix.
11. Centrifuge the sample plate or the tube at 280 × g for 30 seconds.