- 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
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
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- PromethION 2 Solo device
- PromethION 24/48 device
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
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Using the Library Solution
We recommend using the Library Beads (LIB) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Library Solution (LIS) instead of water.
Note: Some customers have noticed that viscous libraries can be loaded more easily when not using Library Beads (LIB). -
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 before storing on ice.
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Prepare the flow cell priming mix in a suitable tube for the number of flow cells to flush. Once combined, mix well by briefly vortexing.
Reagents 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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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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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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Optional actionThe Multiplex Ligation Kit V14 XL is designed for users running multiple flow cells. When handling multiple DNA libraries, the Sequencing Buffer (SB) and Library Beads (LIB) can be combined in a master mix:
- Mix the Sequencing Buffer (SB) and Library Beads (LIB) 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 168 µl of the master mix into each DNA sample-containing tube.
- Mix the samples 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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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 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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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.
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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.