- Materials
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- 12 µl of adapted DNA library (from previous step)
- Flow Cell Wash Kit (EXP-WSH004)
- Flow cell priming reagents available in your sequencing kit or in the following kits:
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Flow Cell Priming Kit V14 (EXP-FLP004)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- P1000 pipette and tips
- P20 pipette and tips
- Ice bucket with ice
- Vortex mixer
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We recommend washing and reloading the flow cell after ~24 hours of sequencing.
We recommend washing and reloading the flow cell after ~24 hours of sequencing.
For this method, the flow cell is washed after ~24 hours of sequencing to restore pores to ensure efficient data acquisition. After an additional 24 hours of sequencing, the flow cell is washed and reloaded a second time.
For this reason, enough library was generated for 3 flow cell loads in the adapter ligation step of the protocol.- This washing procedure aims to remove most of the initial library and unblock the pores to prepare the flow cell for the loading of a subsequent library.
- Data acquisition in MinKNOW should be paused during the wash procedure and library loading.
- After the flow cell has been washed, the next library can be loaded.
You can navigate to the Pore Activity or the Pore Scan Results plot to see pore availability.
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Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
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Thaw one tube of Wash Diluent (DIL) at room temperature.
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Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.
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In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the following Flow Cell Wash Mix:
Reagent Volume per flow cell Wash Mix (WMX) 2 μl Wash Diluent (DIL) 398 μl Total 400 μl -
Mix well by pipetting, and place on ice. Do not vortex the tube.
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Pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
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Before removing the waste fluid, ensure that the flow cell priming port cover and SpotON sample port cover are closed, as indicated in the figure below.
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Remove all fluid from the waste channel through waste port 1 using a P1000 pipette.
As both the flow cell priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
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Slide the flow cell priming port cover clockwise to open.
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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 flow cell priming port.
- Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer/liquid entering the pipette tip.
- Visually check that there is continuous buffer from the flow cell priming port across the sensor array.
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Slowly load 200 µl of the prepared flow cell wash mix into the priming port, as follows:
- Using a P1000 pipette, take 200 µl of the flow cell wash mix
- Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
- Set a timer for a 5 minute incubation.
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Once the 5 minute incubation is complete, carefully load the remaining 200 µl of the prepared flow cell wash mix into the priming port, as follows:
- Using a P1000 pipette, take the remaining 200 µl of the flow cell wash mix
- Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
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Close the priming port and wait for 1 hour.
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Before removing the waste fluid a second time, ensure that the flow cell priming port cover and SpotON sample port cover are closed, as indicated in the figure below.
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Remove all fluid from the waste channel through waste port 1 using a P1000 pipette.
As both the flow cell priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
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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 before storing on ice.
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To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at room temperature.
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.
Single-use tubes format:
Add 5 µl Bovine Serum Albumin (BSA) at 50 mg/ml and 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).Bottle format:
In a suitable tube for the number of flow cells, combine the following reagents:Reagent Volume per flow cell Flow Cell Flush (FCF) 1,170 µl Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl Flow Cell Tether (FCT) 30 µl Total volume 1,205 µl -
Slide the 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 flow cell priming port.
- Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer/liquid entering the pipette tip.
- Visually check that there is continuous buffer from the flow cell 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 via the 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, close the priming port and replace the MinION or GridION device lid.
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Select "Start pore scan" before resuming sequencing to check recovery rate of pores.
Allow the pore scan to complete and ensure sufficient pores are available to continue sequencing.
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Select "Resume run" to continue the sequencing run.