<aside> 💡 For library preparation, please see Library Prep. This page describes how to set up and load the iSeq.

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https://www.protocols.io/view/iseq-bacterial-wgs-protocol-5qpvo5zbxl4o/v1

Preparation of Cartridge

Wear powder-free gloves. Thaw by following one of three ways listed below. Prior to thawing, remove the cartridge from the box but do not open the white foil bag! After thawing, do not refreeze. Store cartridge at -15˚C to -25˚C at least one day before thawing using any of the methods listed below.

Dilution of 1 nM Library to Loading

1. Remove a new iSeq flow cell from 2˚C to 8˚C storage and set aside the unopened package at room temperature for 10-15 minutes. This prevents condensation and ensures that it reaches room temperature on time.
2. Combine 5 μL of 1 nM libray with 95 μL of RSB (or 10 mM Tris-HCl, pH 8.5) in a low-bind microtube to a loading concentration of 50 pM.
3. Vortex briefly, and then centrifuge at 800 – 1200 rpm (or 280 x g) for 1 minute. Place on ice.
4. Thaw the 10 nM PhiX stock at room temperature for 10 minutes.
5. Vortex and pulse spin the thawed 10 nM PhiX stock.
6. Combine 5 μL of 10 nM PhiX stock with 45 μL RSB in a low-bind microtube to prepare 50 μL of 1 nM PhiX. Vortex briefly, and then centrifuge at 800 – 1200 rpm (or 280 g) for 1 minute. Label the prepared 1 nM PhiX tube, which can be stored at -25˚C to -15C˚ for up to 1 month and re-used within this time period.
7. Further dilute PhiX to 50 pM: Combine 5 μL 1 nM PhiX with 95 μL RSB in a low-bind microtube.
8. Vortex briefly, and then centrifuge at 800 – 1200 rpm (or 280 x g) for 1 minute. Place on ice.
9. Add 2 μL of 50 pM PhiX to 100 μL of 50 pM library.
10. Vortex briefly, and then centrifuge at 800 – 1200 rpm (or 280 x g) for 1 minute. Set aside the library with PhiX spike-in on ice.

Load the flow cell

1. Open the cartridge bag from the notches.