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When to refuel
If the DNA translocation speed drops below 300 bases per second, you may start to see a reduction in quality of data reflected in the Qscore. We therefore recommend topping up the flow cell with fuel, using the Flush Buffer (FB) from the Flow Cell Priming Kit. Please follow the instructions below if you wish to top up the fuel during an experiment.
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Refuelling a Flongle™ flow cell
- Remove one tube of FB from the freezer and thaw by bringing to room temperature
Pause the experiment on the GridION position/on the MinION Mk 1B Flow Cell that is being refuelled:
a. Navigate to Experiments and open the experiment running
b. Click Pause
c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm
- Pull back the seal tab to reveal the sample port
- Pass 30 µl of FB through sample port
- Reseal the tab, ensuring the drain ports are covered
- Unpause the experiment on the relevant GridION position/on the MinION Mk 1B by clicking Resume
- (optional) Click Start pore scan to pick a new set of the best channels for the remainder of the sequencing experiment
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Refuelling a MinION™/GridION™ flow cell
Instructions for refuelling a MinION/GridION flow cell:
- Remove one tube of Flush Buffer (FB) from the freezer and thaw by bringing to room temperature
Pause the experiment on the GridION position/on the MinION Mk1B Flow Cell that is being refuelled:
a. Navigate to Experiments and select the experiment running
b. Click Pause
c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm
After opening the priming port, check for a small bubble under the cover. Draw back a small volume to remove the bubble (a few µl):
a. Set a P1000 pipette to 200 µl
b. Insert the tip into the priming port
c. Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip
Visually check that there is continuous buffer from the priming port across the sensor array.
Complete the flow cell refuelling:
- Load 250 µl of the FB into the flow cell via the priming port (i.e. not the SpotON port), avoiding the introduction of air bubbles
- Close the priming port and replace the MinION Mk1B/GridION lid
- Unpause the experiment on the relevant GridION position/on the MinION Mk1B by clicking Resume
- (optional) Click Start pore scan and choose your flow cell, to pick a new set of the best channels for the remainder of the sequencing experiment
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Refuelling a PromethION™ flow cell
- Remove one tube of FB from the freezer and thaw by bringing to room temperature
- Open the valve to reveal the inlet port
Pause the experiment on the relevant PromethION Flow Cell position(s) that will be refuelled:
a. Navigate to Experiments and open the experiment running
b. Click Pause
c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm
- Set a P1000 pipette to 200 µl
- Insert the tip into the inlet port
- Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.
- Using a P1000 pipette, load 250 µl of the FB into the inlet port of the flow cell, avoiding the introduction of air bubbles
- Close the valve
- Incubate the flow cell with the added FB for 3 mins
- Unpause the experiment on the relevant PromethION Flow Cell position(s) after this process is complete by clicking Resume
- (optional) Click Start pore scan and choose your flow cell, to pick a new set of the best channels for the remainder of the sequencing experiment
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The outcome and benefits of refuelling
Translocation speed and Qscore over time
Below is a graph that shows what is expected for translocation speed after the addition of FB to the flow cell in a previous version of MinKNOW.
As the speed drops below 300 bases per second, the Qscore begins to decline for the reads processed through the nanopores at this speed. After refuelling at the 17.5 hour mark, the speed begins to increase and returns to an improved rate (~400 bases per second), which is similar to the speed at the start of the experiment. After the addition of fuel, the quality of the data increases and returns to Qscores equivalent to those seen at the start of the run.
Refuelling multiple times in a run
You can refuel a sequencing run multiple times over an experiment. When you should refuel will depend on when the translocation speed drops below 300 bases per second on the speed graph in the MinKNOW GUI for Kit 9 (e.g. SQK-LSK109). Kit 14 (e.g. SQK-LSK114) adapter contains fuel fix technology, enabling users to run long experiments without the need for fuel addition.