- Materials
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- Sequencing Buffer (SB)
- Library Beads (LIB)
- Library Solution (LIS)
- Flow Cell Tether (FCT)
- Flow Cell Flush (FCF)
- Consumables
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- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- PromethION device
- PromethION Flow Cell Light Shield
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
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Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
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To prepare the flow cell priming mix, combine Flow Cell Tether (FCT) and Flow Cell Flush (FCF), as directed below. Mix by vortexing at room temperature.
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.
Single-use tubes format:
Add 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).Bottle format:
In a clean suitable tube for the number of flow cells, combine the following reagents:Reagent Volume per flow cell Flow Cell Flush (FCF) 1,170 µl Flow Cell Tether (FCT) 30 µl Total volume 1,200 µl -
For PromethION 2 Solo, load the flow cell(s) as follows:
Place the flow cell flat on the metal plate.
Slide the flow cell into the docking port until the gold pins or green board cannot be seen.
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For the PromethION 24/48, load the flow cell(s) into the docking ports:
- Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
- Press down firmly onto the flow cell and ensure the latch engages and clicks into place.
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Slide the inlet port cover clockwise to open.
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After opening the inlet port, draw back a small volume to remove any air bubbles:
- Set a P1000 pipette tip to 200 µl.
- Insert the tip into the inlet port.
- Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.
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Load 500 µl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes. During this time, prepare the library for loading using the next steps in the protocol.
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Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
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In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
Reagent Volume per flow cell Sequencing Buffer (SB) 100 µl Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS) 68 µl DNA library 32 µl Total 200 µl Note: Library loading volume has been increased to improve array coverage.
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Complete the flow cell priming by slowly loading 500 µl of the priming mix into the inlet port.
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Mix the prepared library gently by pipetting up and down just prior to loading.
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Load 200 µl of library into the inlet port using a P1000 pipette.
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Close the valve to seal the inlet port.
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If the light shield has been removed from the flow cell, install the light shield as follows:
- Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
- Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.