Loading and Running a Flow Cell on a Oxford Nanopore Technologies MinION

Materials and Equipment

Flow cell & device

• MinION device (or compatible device)
• FLO-MIN114 flow cell (R10.4.1)
• Flow cell light shield (for MinION)

Reagents (for loading/priming)

• Flow Cell Flush (FCF)
• Flow Cell Tether (FCT)
• Sequencing Buffer (SB)
• Library Beads (LIB) or Library Solution (LIS)
• Bovine Serum Albumin (BSA) 50 mg/mL (for priming mix) ([Oxford Nanopore Technologies][1])
• Prepared DNA library (from your library prep kit)
• Nuclease-free water

Consumables & equipment

• 1.5 mL Eppendorf DNA LoBind tubes
• Pipettes: P1000, P200, P20, P10 (with filtered tips)
• Microfuge or centrifuge for brief spin-down
• Timer
• Thermal cycler or heat block (if library prep needs incubation)
• Ice bucket
• Optional QC equipment: Qubit fluorometer, Nanodrop, etc.
• Good laboratory practice: gloves, lab coat, sterile tips, aerosol barrier tips

Pre-run Checklist

1. Ensure flow cell has been stored and handled according to manufacturer guidelines (e.g. correct temperature).
2. Inspect flow cell for shipping damage.
3. Perform a Flow Cell Check (via MinKNOW) to verify active pore count. ONT recommends for R10.4.1 flow cells: check before the run. ([Oxford Nanopore Technologies][2])
4. Ensure your library prep is completed and you have the correct amount of library (for time-saving you may start priming while doing final library steps).
5. Bring flow cell to room temperature for ~20 minutes (per Rapid Barcoding V14 protocol) before loading. ([Oxford Nanopore Technologies][2])
6. Have all reagents thawed mixed and on ice as appropriate.

Insert flow cell into device

1. Open the lid of the MinION.
2. Slide the flow cell under the clip (for the device) and press down firmly on the priming port cover to ensure thermal/electrical contact. ([Oxford Nanopore Technologies][2])
3. Ensure the priming port cover is closed until you are ready for priming.

Prepare priming mix

1. Thaw FCF, FCT, SB (and LIB/LIS if required) at room temperature. Then spin down and put on ice. ([Oxford Nanopore Technologies][1])

2. Prepare priming mix with BSA: For FLO-MIN114, combine:

   • FCF: 1,170 µL
   • BSA 50 mg/mL: 5 µL (final ≈0.2 mg/mL) ([Oxford Nanopore Technologies][1])
   • FCT: 30 µL – Mix by pipetting gently. ([Oxford Nanopore Technologies][2])

Remove potential air bubble and prime flow cell

1. Open the priming port cover by sliding it clockwise. ([Oxford Nanopore Technologies][2])
2. Check for a small air bubble under the priming port cover. Use a P1000 pipette set to ~200 µL, insert tip into priming port, withdraw 20-30 µL (set wheel to ~220-230 µL) until a small volume enters the tip. Ensure continuous buffer covers the pore array. Do not remove more than ~20-30 µL. ([Oxford Nanopore Technologies][1])
3. Load 800 µL of the priming mix into the priming port, avoiding air bubbles. Wait 5 minutes. ([Oxford Nanopore Technologies][1])

Prepare library for loading

1. Mix LIB (if using beads) immediately before use (they settle quickly). ([Oxford Nanopore Technologies][1])

2. In a clean 1.5 mL tube, prepare mixture:

   • Sequencing Buffer (SB): 37.5 µL ([Oxford Nanopore Technologies][2])
   • Library Beads (LIB) or Library Solution (LIS): if using LIB, 25.5 µL; if LIS use 12 µL (per alternative) ([Oxford Nanopore Technologies][2])
   • DNA library: 12 µL (or specified amount)
   • Total: ~75 µL

3. Mix gently by pipetting up/down just prior to loading.

Final priming and library loading

1. Lift the SpotON sample port cover to expose sample port. ([Oxford Nanopore Technologies][1])
2. Load 200 µL of the priming mix into the priming port (not the SpotON port) to complete priming. ([Oxford Nanopore Technologies][2])
3. Drop-wise load 75 µL of the prepared library into the SpotON sample port. Add each drop, let it flow in before next. ([Oxford Nanopore Technologies][1])
4. Replace the SpotON port cover gently, ensure the bung is seated, and close priming port. ([Oxford Nanopore Technologies][2])
5. Place the light shield onto the flow cell for best performance. Ensure it sits properly and avoid forcing it under the clip. ([Oxford Nanopore Technologies][1])
6. Close the device lid. Flow cell is now ready for sequencing.

Initiate sequencing run in MinKNOW

1. Open MinKNOW software and select “Start sequencing”.
2. Enter experiment details: sample ID, flow cell model (FLO-MIN114), kit type.
3. Select the kit used (e.g., Rapid Sequencing Kit V14 or Rapid Barcoding Kit V14). ([Oxford Nanopore Technologies][2])
4. Set output parameters or accept defaults.
5. Click “Start”. Monitor pore occupancy, channel activity, yield.

Post-run and Flow Cell Reuse

• If you intend to reuse the flow cell: perform a wash using the Flow Cell Wash Kit (EXP-WSH004) as soon as possible after run completion. Store at +2 °C to +8 °C if not used immediately. ([Oxford Nanopore Technologies][1])
• If returning to vendor: follow manufacturer’s return procedure.
• Record all relevant run metadata: flow cell ID, pore count at start, library details, sample IDs, run duration, yield.

Safety and Handling Notes

• Avoid introducing air bubbles into the pore array—can irreversibly damage pores. ([Oxford Nanopore Technologies][1])
• Always ensure the flow cell matrix is covered by buffer during priming/loading.
• Use filtered tips; avoid rough pipetting which may damage membrane or pores.
• Dispose of used flow cell, tips, reagents according to lab bio-hazard and chemical waste procedures.
• Adhere to manufacturer’s storage and handling instructions for flow cells and reagents.

Troubleshooting – Common Issues