- Materials
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- Flush Buffer (FB)
- Flush Tether (FLT)
- Loading Beads II (LBII)
- Sequencing Buffer II (SBII)
- Loading Solution (LS)
- Consumables
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- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- PromethION 2 Solo device
- PromethION sequencing device
- PromethION Flow Cell Light Shield
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
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Using the Loading Solution
We recommend using the Loading Beads II (LBII) 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 Loading Solution (LS) instead of water.
Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II. -
Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down the SBII and FLT at room temperature.
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To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing.
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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 Loading Beads II (LBII) by pipetting.
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In a new tube, prepare the library for loading as follows:
Reagent Volume per flow cell Sequencing Buffer II (SBII) 75 µl Loading Beads II (LBII) thoroughly mixed before use, or Loading Solution (LS), if using 51 µl DNA library 24 µl Total 150 µl -
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.
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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.