Hybridize Probes

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EHB2 (Enrichment Hyb Buffer 2)

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NHB2 (Hyb Buffer 2 + IDT NXT Blockers)

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Illumina Viral Surveillance Panel v2 panel

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96-well PCR plate, semiskirted

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Microseal 'B' adhesive film

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NHB2

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Precipitates and separates during storage.

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Follow the preparation instructions before first use.

1.

Preheat the microheating system to 50°C.

3.

Mix preheated NHB2 as follows.

a.

Vortex three times for 10 seconds each time.

b.

Pipette to resuspend fully. Keep warm until use to prevent the reformation of precipitates.

4.

If EHB2 or NHB2 appears crystallized or cloudy, vortex or pipette until clear.

5.

Save the following HYB program on the thermal cycler:

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Choose the preheat lid option and set to 100°C

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Set the reaction volume to 25 µl

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95°C for 5 minutes

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18 cycles of 1 minute each:

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94°C for the first cycle

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Decrease 2°C per subsequent cycle

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58°C for 90 minutes

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Hold at 58°C for ≤ 24 hours

Total running time is ~115 minutes.

1.

Add the following reagents to each well of a new PCR plate in the order listed.

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Three-plex pre-enriched library pool (7.5 µl)

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NHB2 (12.5 µl)

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Illumina Viral Surveillance Panel v2 (2.5 µl)

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EHB2 (2.5 µl)

2.

Pipette 10 times to mix, and then seal.

3.

Centrifuge at 280 × g for 3 seconds.

EHB2 can make the reaction appear cloudy, which is normal.

4.

Place on the preprogrammed thermal cycler and run the HYB program.

5.

Allow to incubate at 58°C for 90 minutes to 24 hours.

Each well contains a volume of 25 µl.