Hybridize Probes

This step binds targeted regions of the DNA with UPIP capture probes.

• EHB2 (Enrichment Hyb Buffer 2)
• UPIP (Urinary Pathogen ID/AMR Panel)
• NHB2 (Hyb Buffer 2 + IDT NXT Blockers)
• RSB (Resuspension Buffer)
• One of the following containers:
– [Plate] 96-well PCR plate
– [Tube] 8-tube strip
• One of the following seals:
– [Plate] Microseal 'B' adhesive seal
– [Tube] 8-tube strip caps
• NHB2 precipitates and separates during storage. Follow the NHB2 preparation instructions before first use.
1. Prepare the following consumables:

Item

Storage

Instructions

EHB2

2°C to 8°C

Bring to room temperature. Vortex to mix.

If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix well until the solution is clear.

UPIP

‑25°C to ‑15°C

Bring to room temperature. Vortex to mix.

Use a total probe volume of 2.5 µl per hybridization reaction.

NHB2 (blue cap)

‑25°C to ‑15°C

Thaw at room temperature.

When at room temperature, preheat to 50°C on a microheating system for 5 minutes.

Vortex at maximum speed three times for 10 seconds each to resuspend.

Centrifuge briefly.

Pipette up and down from the bottom of the tube.

If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix well until the solution is clear.

Use while warm to avoid precipitates from reforming.

SMB3

2°C to 8°C

If you are proceeding to the next procedure immediately after the 90 minute hold in the NF-HYB program, bring to room temperature.

If you are extending the hold time, bring to room temperature at least 30 minutes before the NF-HYB program ends.

EEW (amber tube)

‑25°C to ‑15°C

If you are proceeding to the next procedure immediately after the 90 minute hold in the NF-HYB program, bring to room temperature.
2. Save the following NF-HYB program on the thermal cycler.
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 25 μl
• 95°C for 5 minutes
• 18 cycles of 1 minute each, starting at 94°C for the first cycle, then decreasing 2°C per cycle
• Hold for 90 minutes at 58°C
• [Optional] Extend the 58°C hold to a maximum of 24 hours

Total running time is ~115 minutes.

1. Add the following reagents to each well of a new PCR plate or 8-tube strip in the order listed.

Creating a master mix of NHB2 and EHB2 or failing to add the reagents in the exact order and volumes listed negatively impacts hybridization.

a. Pre-enriched library pool (3-plex) (7.5 μl)
b. NHB2 (blue cap) (12.5 μl)
c. UPIP panel (2.5 μl)
d. EHB2 (2.5 μl)
2. Using a pipette set to 20 µl, pipette each enrichment reaction 10 times to mix.
3. Centrifuge as follows.
• [Plate] Seal the plate with Microseal 'B' and centrifuge at 280 × g for 30 seconds.
• [Tube] Cap the tubes and centrifuge at 280 × g for 30 seconds.
4. Place the sample plate or tube on the preprogrammed thermal cycler and run the NF‑HYB program.
5. Proceed immediately to the next procedure before the NF‑HYB program hold temperature time ends.

Do not allow the hybridization reactions to cool. Precipitation occurs if the temperature of the hybridization reaction falls below room temperature.