- Materials
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- Flow Cell Wash Kit (EXP-WSH004) or Flow Cell Wash Kit XL (EXP-WSH004-XL)
- Flow cell priming reagents available in your sequencing kit or in the following kits:
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Flow Cell Priming Kit (EXP-FLP004)
- Equipment
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- P1000 pipette and tips
- P20 pipette and tips
- Ice bucket with ice
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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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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.
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 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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Slowly load 800 µl of the priming mix into the priming port, as follows:
- Using a P1000 pipette, take 800 µl of the priming 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, as illustrated in the video above, leaving a small volume of buffer in the pipette tip.
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Close the priming port and wait 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 tube, prepare the library for loading according to the "Priming and loading the MinION and GridION Flow Cell" section of the suitable protocol to ensure you are using the correct reagents and volumes.
For Kit 14 chemistry:
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 Recovered DNA library 12 µl Total 75 µl -
Remove the waste buffer, as follows:
- Ensure the priming port and SpotON sample port covers are closed, as indicated in the figure below.
- Insert a P1000 pipette into waste port 1 and remove the waste buffer.
Note: As both the 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 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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Slowly load 200 µl of the priming mix into the flow cell priming port, as follows:
- Ensure the priming port is open and gently lift open the SpotON sample port.
- Using a P1000 pipette, take 200 µl of the priming 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, as illustrated in the video above, leaving a small volume of buffer in the pipette tip.
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Remove the waste buffer, as follows:
- Close the priming port and SpotON sample port cover, as indicated in the figure below.
- Insert a P1000 pipette into waste port 1 and remove the waste buffer.
Note: As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
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Slide open the priming port cover and gently lift open the SpotON sample port cover.
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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.