- Materials
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- 10 ng high molecular weight genomic DNA
- 16S Barcoding Kit 24 V14 (SQK-16S114.24)
- Consumables
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- MinION and GridION Flow Cell
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes
- Equipment
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- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- Hula mixer (gentle rotator mixer)
- Microfuge
- Vortex mixer
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent for QC check)
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For this protocol, you will need 10 ng high molecular weight genomic DNA per barcode.
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Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
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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 -
16S Barcoding Kit 24 V14 contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) 16S Barcode Primers 01-24 1-24 - 2 plates, 3 sets of barcodes per plate 15 μl per well Rapid Adapter RA Green 1 15 Adapter Buffer ADB Clear 1 100 AMPure XP Beads AXP Clear cap, light teal label 1 6,000 Elution buffer EB Black 1 1,500 EDTA EDTA Blue 1 700 Sequencing Buffer SB Red 1 700 Library Beads LIB Pink 1 600 Library Solution LIS White cap, pink label 1 600 Flow Cell Flush FCF Clear cap, light blue label 1 8,000 Flow Cell Tether FCT Purple 1 200 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.