- Materials
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- 200 ng of extracted gDNA per sample
- 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
- Monarch® Genomic DNA Purification Kit (NEB, T3010S/L)
- Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
- Qubit 1x dsDNA BR Assay Kit (ThermoFisher, cat # Q33265)
- PowerBead Pro tube (Qiagen, 19301)
- BashingBead Buffer (Zymo, D6001-3-40)
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- TE buffer (Sigma, 8890-100ML)
- 5 M sodium chloride solution (Sigma, S6546)
- CTAB buffer (Promega, MC1411)
- (Optional) Extra reagents for custom CTAB buffer:
- CTAB (Sigma, H6269)
- 0.5 M EDTA (Fisher Scientific, 11568896)
- Trizma® hydrochloride solution (Sigma, T2819)
- Extra reagents for bacteria gDNA extraction:
- Lysozyme human (Sigma, L1667)
- Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
- Trizma® hydrochloride solution (Sigma, T2819)
- Achromopeptidase (Sigma, A3547)
- Extra reagents and consumables for hard to lyse organisms gDNA extraction:
- Lysozyme human (Sigma, L1667)
- Empty FastPrep® 2mL Lysing Matrix tubes (MP Biomedicals, 115076200)
- Screw Cap for 2 mL Lysing Matrix tubes (MP Biomedicals, 115067005)
- Glass beads, 4 mm (MP Biomedicals, 116914801)
- Extra reagents for fungi gDNA extraction:
- MetaPolyzyme (Sigma, MAC4L-5MG)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- Extra reagents for automated bead-beating gDNA extraction:
- RNase A (QIAGEN, 19101)
- Proteinase K (QIAGEN, 19131)
- Maxwell® RSC PureFood Pathogen kit (Promega, AS1660)
- 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)
- PCR plate seals
- 96-well PCR plate, semi-skirted (e.g. Starlab, I1402-9800)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- Vortex Adapter (Qiagen 13000-V1-24)
- Vortex mixer
- Thermal cycler
- Timer
- Thermomixer
- Magnetic rack
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Eppendorf 5424 centrifuge (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Multichannel pipette and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Extra equipment for automated bead-beating gDNA extraction:
- Maxwell® RSC Instrument (MPBiomedicals, AS4500)
- Optional equipment
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- Hula mixer (gentle rotator mixer)
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For this protocol, the following inputs are required per sample:
Input requirements per sample for the extraction methods:
Universal bead-beating gDNA extraction: 1 ml liquid overnight culture (~1 x 108 – 109 cfu/ml) or half of a loop of colonies from a plate
Bacteria gDNA extraction: 200 µl liquid overnight culture (~1 x 108 – 109 cfu/ml) or 1/8 of a loop of colonies from a plate
Hard to lyse organisms gDNA extraction: 5 – 10 mg cells from solid or liquid media
Fungi gDNA extraction: 2 ml of ~1 x 107 cfu/ml overnight culture or a full 10 µl inoculating loop from a plate
For library preparation, 200 ng in 10 µl of extracted gDNA per sample is required.
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Third-party reagents
Depending on the extraction protocol used, not all third-party reagents are required.
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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Custom CTAB buffer
Custom CTAB buffer can be prepared instead of purchasing. Below are the reagents and concentrations required, with suggested examples with excess.
Reagent Stock Final concentration Volume for 12 samples Volume for 24 samples CTAB - 2% v/v 60 µl 120 µl EDTA 0.5 M 40 mM 240 µl 480 µl Sodium chloride 5 M 1.4 M 833 µl 1,666 µl Trizma hydrochloride solution, pH 8 1 M 100 mM 300 µl 600 µl Nuclease-free water - - 1567 µl 3,134 µl Total - - 3,000 µl 6,000 µl -
For staphylococcal inputs
Staphylococcal Lysis Buffer (SLB) is required for the bacterial gDNA extraction method for staphylococcal inputs.
Reagent Stock Final concentration Volume for 12 samples with excess Volume for 24 samples with excess Trizma hydrochloride solution, pH 9 1 M 100 mM 150 ul 300 µl Sodium chloride 5 M 10 mM 3 ul 6 µl SDS 10% v/v 0.1% v/v 15 ul 30 µl Nuclease-free water - - 1332 ul 2664 µl Total volume - - 1,500 ul 3,000 µl The SDS in the Staphylococcal Lysis Buffer (SLB) is essential for preventing the degradation of staphylococci DNA. Exclusion of SDS from the buffer results in a larger smear of DNA when run on a gel.
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Input DNA
How to QC your input DNA
It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.
Chemical contaminants
Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.
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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
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Rapid Adapter RA Green 1 15 Adapter Buffer ADB Clear 1 100 AMPure XP Beads AXP Amber 2 1,200 Elution Buffer EB Black 1 500 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 Blue 6 1,170 Flow Cell Tether FCT Purple 1 200 Rapid Barcodes RB01-24 Clear 24 15 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
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Rapid Adapter RA Green 2 15 Adapter Buffer ADB Clear 1 100 AMPure XP Beads AXP Amber 3 1,200 Elution Buffer EB Black 1 1,500 Sequencing Buffer SB Red 1 1,700 Library Beads LIB Pink 1 1,800 Library Solution LIS White cap, pink label 1 1,800 Flow Cell Flush FCF Clear 1 15,500 Flow Cell Tether FCT Purple 2 200 Rapid Barcodes RB01-96 - 3 plates 8 µl per well 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.