- Materials
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- 50 ng amplicon DNA per sample (500 bp – 5 kb amplicon size)
- Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) OR Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
- Consumables
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- MinION and GridION Flow Cell
- Agencourt AMPure XP beads (Beckman Coulter™, A63881) (or equivalent for DNA purification)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
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- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- Hula mixer (gentle rotator mixer)
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- Vortex mixer
- Thermal cycler or heat blocks
- Qubit fluorometer (or equivalent for QC check)
- Magnetic rack
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
- Ice bucket with ice
- Timer
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For this protocol, you will need 50 ng of single species amplicon DNA per sample.
We recommend performing a purification step following the PCR reaction used to generate your amplicons. Please use standard methods appropriate for the amplicon size, such as AMPure XP beads. This step ensures the removal of proteins, salts, dNTPs, and primers, which could potentially impact the library preparation and subsequent analysis workflow.
Please note that while alternative methods exist, they have not been validated by our internal teams.
This method is intended for barcoding single species amplicon samples and is not suitable for mixed species amplicons.
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Check your flow cell
We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within three months of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.
Flow cell Minimum number of active pores covered by warranty Flongle Flow Cell 50 MinION/GridION Flow Cell 800 PromethION Flow Cell 5000 -
Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) contents
Single-use tubes format:
Bottle format:
Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
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Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) contents
Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.