- Materials
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- Sequencing Buffer (SQB)
- Loading Beads (LB)
- Flow Cell Priming Kit XL (EXP-FLP002-XL)
- Consumables
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- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
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- PromethION 2 Solo device
- PromethION sequencing device
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
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Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and Flush Buffer (FB) at room temperature before mixing the reagents by vortexing and spin down.
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Prepare the flow cell priming mix in a suitable vial for the number of flow cells to flush. Once combined, mix well by briefly vortexing.
Reagent Volume per flow cell Flush Tether (FLT) 30 µl Flush Buffer (FB) 1,170 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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If not already completed, perform a flow cell check on all flow cells.
Please refer to the Flow Cell Check protocol for further information.
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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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Meanwhile, thoroughly mix the contents of the thawed Sequencing Buffer (SQB) and Loading Beads (LB) tube(s) by vortexing.
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In a separate tube for each library, prepare for loading by adding the following reagents:
Reagent Volume SQB 75 µl LB 51 µl DNA library 24 µl Total 150 µl -
Optional actionThe Ligation Sequencing Kit XL kit is designed for users running multiple samples/flow cells. When handling multiple DNA preparations, the Sequencing Buffer (SQB) and Loading Beads (LB) can be combined in a master mix:
- Mix the Sequencing Buffer (SQB) and Loading Beads (LB) as described above, scaling up the final volume for the appropriate number of samples and adding up to 20% excess of each reagent.
- Mix the master mix by pipetting immediately before adding to the DNA samples.
- Pipette 126 µl of the master mix into each DNA sample-containing tube.
- Mix the sample by pipetting.
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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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Using a P1000, insert the pipette tip into the inlet port and add 150 µl of library.
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Close the valve to seal the inlet port and close the PromethION lid when ready.
Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.
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For multiple flow cell washing, use the same experiment name and identifying sample IDs for all runs to enable all flow cells to be paused simultaneously.