- Materials
-
- Flush Buffer (FB)
- Flush Tether (FLT)
- Consumables
-
- MinION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
-
- P1000 pipette and tips
- P100 pipette and tips
-
Open the MinION Mk1B lid and slide the flow cell under the clip.
Press down firmly on the flow cell to ensure correct thermal and electrical contact.
-
Optional actionComplete a flow cell check to assess the number of pores available before loading the library.
This step can be omitted if the flow cell has been checked previously.
See the flow cell check instructions in the MinKNOW protocol for more information.
-
Slide the priming port cover clockwise to open the priming port.
-
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few µls).
- Set a P1000 pipette to 200 µl
- Insert the tip into the priming port
- Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, 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.
-
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
-
To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.
-
Thoroughly mix the contents of the VX Running Buffer (VRB) tube by pipetting up and down.
-
In a new tube, prepare the library for loading as follows:
Reagent Volume per flow cell VX Running Buffer (VRB) 64 µl DNA library 11 µl Total 75 µl -
Complete the flow cell priming:
- Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
- Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
-
Mix the prepared library gently by pipetting up and down just prior to loading.
-
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.
-
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and close the lid of the sequencing device.