- Materials
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- Flow Cell Flush (FCF)
- Flow Cell Tether (FCT)
- Library Solution (LIS)
- Library Beads (LIB)
- Sequencing Buffer (SB)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Equipment
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- MinION or GridION device
- SpotON Flow Cell
- MinION and GridION Flow Cell Light Shield
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
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Using the Library Solution
For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).
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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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Prepare the flow cell priming mix with BSA in a suitable vial for the number of flow cells to flush. Once combined, mix well by inverting the tube and pipette mix at room temperature.
Reagent Volume per flow cell Flow Cell Tether (FCT) 30 µl Flow Cell Flush (FCF) 1170 µl Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl Total 1,205 µl -
Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.
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Optional actionComplete a flow cell check to assess the number of pores available before loading the library.
This step can be omitted if the flow cell has been checked previously.
See the flow cell check instructions in the MinKNOW protocol for more information.
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Slide the flow cell priming port cover clockwise to open the priming port.
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After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
- Set a P1000 pipette to 200 µl
- Insert the tip into the priming port
- Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip
Note: Visually check that there is continuous buffer from the priming port across the sensor array.
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Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
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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) 37.5 µl Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using 25.5 µl DNA library 12 µl Total 75 µl -
Complete the flow cell priming:
- Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
- Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
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Mix the prepared library gently by pipetting up and down just prior to loading.
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Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
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Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
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Place the light shield onto the flow cell, as follows:
Carefully place the leading edge of the light shield against the clip.
Note: Do not force the light shield underneath the clip.Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.