- 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 (FLO-PRO114M)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- PromethION 24/48 device
- PromethION Flow Cell Light Shield
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
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Day 1: Duplex experiment flow cell loading
Once the library has been prepared, the PromethION Flow Cell can be primed before the library is combined with the sequencing reagents and loaded into the flow cell.
We recommend monitoring your sequencing run and to reload your flow cell when recommended to in the "Washing and reloading Duplex library on PromethION Flow Cells". The Duplex experiment can be reloaded three times, across days 2, 3 and 4.
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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 and store 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 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.