Priming MinION flow cell

[jalinaodb]

Hi!

I've been working with the MinION from Oxford Nanopore for a small project. I used the Rapid Sequencing Kit (RAD002) and followed the protocol from the Rapid Lambda Control Experiment. I've been going through the protocol step by step, trying to understand all the steps. Most of them I've figured out. One step however I don't fully understand: Priming the flow cell.

Before loading your library you need to prepare a priming mix, containing RBF and nuclease-free water. First you load an amount into the priming port, then you load some into the flow cell via the SpotOn port. Why is this necessary? What exactly does this priming mix do?

Another question: are the Library Loading Beads really necessary? Or can you get the same results without them?

Hope someone can help!

[gringer]

What exactly does this priming mix do?

I can't give you an exact answer, but my understanding is that the priming mix includes ATP and additional chemicals that improve the rate of sequencing. The second flush of the priming mix (with the SpotON port open) helps to clear any blockages near the SpotON port and generate a bit of suction to help with the drip loading -- the closer this is done to sample loading the better.

are the Library Loading Beads really necessary? Or can you get the same results without them?

Loading beads bring the DNA closer to the pores prior to the start of sequencing. The loading beads aren't necessary for sequencing to be carried out, but they are necessary for high-yield (and high-speed) sequencing. The same results will not be achieved without library loading beads.