On Thursday, we received our Oxford Nanopore Technologies (ONT) P2! I am also starting to watch London Calling 2023 sessions on YouTube. As I am traveling to my first Gordon Research Conference (GRC) tomorrow and I am eager to use our P2, I watched the London Calling 2023 session entitled “How to load a PromethION Flow Cell.” This session was part of the Master Class series. The speaker mentioned that the PromethION flow cells can produce 100s of Gbases! The P2 and P2 solo can handle two flow cells at a time, while the PromethION 24 and 48 can sequence using up to 48 flow cells. Several kits are available and allow for multiplexing. The PromethION flow cell is square and has an inlet, a valve to prevent evaporation, and a waste channel. As with MinION flow cells, the flow cells are shipped with a yellow buffer. The speaker recommended removing flow cells from the refrigerator ~20 minutes before using to prevent condensation from forming on the pins. To insert the flow cell into the P2, the connector pins must line up before “clicking into place.” To begin loading, the inlet valve is opened by turning ninety degrees. A pipette is set to 200 microliters and used to remove the air pocket and a small amount of buffer. The pipette is then set to 500 microliters of Flush Buffer making sure there is no air in the inlet port. They recommend leaving a small amount of liquid in the tip to prevent introduction of air. After five minutes, 500 microliters are added to the inlet port a second time. The second flush results in liquid in the channel. A P200 is set to 200 microliters to introduce the library into the inlet port. The speaker suggested pipetting up and down to resuspend the loading beads before aspirating. They recommend waiting ten minutes after loading before starting the sequencing experiment to stabilize temperature. The session also had video demonstrations of loading a MinION and Flongle flow cells. What caught my attention was that for Flongle flow cell loading, they insert the tip directly into the port. This session was useful to watch carefully what ONT experts do while loading Flongle, MinION, and PromethION flow cells.
