Prime MinION flowcell and load the library
with lots of notes drawn from experience making mistakes
protocol assumes a R9.4 spot on flowcell
Ensure you’ve aleady QC’d the flowcell to determine the number of active pores.
Because the priming process takes a bit of time, you can do the priming steps during incubation periods earlier on in the protocol.
Prepare the priming solution by mixing 576 uL of RBF with 624 uL of nuclease-free water. Mix by inversion and spin down. use the same pipette to ensure that the ratio is correct even if there is error in the pipette.
Open the sample port on the flowcell (leave the SpotON port closed). Draw back a few uL of buffer to ensure there’s fluid up to the lip of the sample port. this is to prevent you from introducing bubbles when priming the flowcell. This is easiest to do by inserting the tip of the pipette into the port and rather than drawing up volume using your normal pipetting action, simply adjust the pipette to increase the volume it holds, which very slowly allows you to draw up the buffer, ensuring you don’t take up too much. This will also prevent shakes where you might accidentally push more buffer than you drew up into the flowcell, introducing a bubble.
Load 500 uL of priming solution via the sample port. Wait 10 minutes. again, it’s more accident proof to load this by adjusting the pipette volume down rather than using a normal pipetting push down. Leave a few uL of priming solution in pipette tip to prevent introduction of a bubble.
Load 300 uL of priming solution via the sample port. Wait 10 minutes.
Lift the SpotON tab to reveal the SpotON port. Load 200 uL of priming solution via the sample port. you should see some fluid come up through the spot on port and then go back down again, this is good and will ensure that the capillary action is working.
- Load the whole library+RBF mixture via the SpotON port drop by drop. To do so, push a droplet to the end of your pipette tip, then touch the droplet to the top of the SpotON port. The capillary action should pull the droplet down into the flowcell. IMPORTANT: sometimes the capillary action doesn’t work, and your first droplet of library just sits on the SpotON port without getting sucked in. If this happens don’t panic, just make up another 200 uL of priming solution and repeat your 200 uL prime via the sample port. Then try loading your library again.
Close the sample port and SpotON port, making sure the bung goes into the SpotON port hole. Close the MinION lid.
- In MinKNOW choose the MAP_48Hr_Sequencing_Run_SQK_LSK108FLOMIN106.py program. _Frustratingly there are pretty regular updates for MinKNOW and the autoupdate function doesn’t seem to work on Mac. So if you’re using a Mac as your sequencing laptop you’ll probably need to do a complete uninstall and reinstall of MinKNOW each time there is an update. Also, if the MinKNOW run isn’t working once you’ve started the sequencing run (e.g. performs calibration but never moves along to sequencing) try uninstalling and reinstalling MinKNOW and restarting the run.