- Materials
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- Native Barcoding Kit 24 (SQK-NBD112.24)
- 400 ng gDNA per sample for >4 barcodes
- 1000 ng gDNA per sample for ≤4 barcodes
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Microfuge
- Magnetic rack
- Vortex mixer
- Thermal cycler
- Multichannel pipette and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
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For this protocol, you will need 400 ng gDNA per sample for >4 barcodes or 1000 ng gDNA per sample for ≤4 barcodes.
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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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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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Native Barcoding Kit 24 (SQK-NBD112.24) contents
Name Acronym Cap colour Number of vials Fill volume per vial (μl) Adapter Mix II H AMII H Green 1 40 Sequencing Buffer II SBII Red 1 500 Loading Beads II LBII Pink 1 360 Loading Solution LS White cap, pink label 1 400 Elution Buffer EB Black 1 200 AMPure XP Beads AXP Amber 3 1,200 Long Fragment Buffer LFB Orange 1 1,800 Short Fragment Buffer SFB Clear 3 1,800 Flush Buffer FB Blue 6 1,170 Flush Tether FLT White cap, purple label 1 200 EDTA EDTA Clear 1 700 Native Barcodes 01-24 NB01-24 Clear 24 20 μl per well 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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The Native Barcoding Expansion kit (EXP-NBD112) is available to provide enough reagents for 12 reactions depending on how barcodes are used.
This kit contains reagents used in the Native Barcoding Kit 24 (SQK-NBD112.24) and the Native Barcoding Kit 96 (SQK-NBD112.96).
Native Barcoding Expansion Kit (EXP-NBD112) contents:
Reagent Acronym Cap colour No. of vials Fill volume per vial (µl) Adapter II H AMII H Green 2 40 AMPure XP Beads AXP Amber 6 1,200 EDTA EDTA Clear 2 700 Long Fragment Buffer LFB Orange 2 1,800 Flush Buffer FB Bottle 1 15,500 Flush Tether FLT Purple 1 400 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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Native barcode sequences
Component Forward sequence Reverse sequence NB01 CACAAAGACACCGACAACTTTCTT AAGAAAGTTGTCGGTGTCTTTGTG NB02 ACAGACGACTACAAACGGAATCGA TCGATTCCGTTTGTAGTCGTCTGT NB03 CCTGGTAACTGGGACACAAGACTC GAGTCTTGTGTCCCAGTTACCAGG NB04 TAGGGAAACACGATAGAATCCGAA TTCGGATTCTATCGTGTTTCCCTA NB05 AAGGTTACACAAACCCTGGACAAG CTTGTCCAGGGTTTGTGTAACCTT NB06 GACTACTTTCTGCCTTTGCGAGAA TTCTCGCAAAGGCAGAAAGTAGTC NB07 AAGGATTCATTCCCACGGTAACAC GTGTTACCGTGGGAATGAATCCTT NB08 ACGTAACTTGGTTTGTTCCCTGAA TTCAGGGAACAAACCAAGTTACGT NB09 AACCAAGACTCGCTGTGCCTAGTT AACTAGGCACAGCGAGTCTTGGTT NB10 GAGAGGACAAAGGTTTCAACGCTT AAGCGTTGAAACCTTTGTCCTCTC NB11 TCCATTCCCTCCGATAGATGAAAC GTTTCATCTATCGGAGGGAATGGA NB12 TCCGATTCTGCTTCTTTCTACCTG CAGGTAGAAAGAAGCAGAATCGGA NB13 AGAACGACTTCCATACTCGTGTGA TCACACGAGTATGGAAGTCGTTCT NB14 AACGAGTCTCTTGGGACCCATAGA TCTATGGGTCCCAAGAGACTCGTT NB15 AGGTCTACCTCGCTAACACCACTG CAGTGGTGTTAGCGAGGTAGACCT NB16 CGTCAACTGACAGTGGTTCGTACT AGTACGAACCACTGTCAGTTGACG NB17 ACCCTCCAGGAAAGTACCTCTGAT ATCAGAGGTACTTTCCTGGAGGGT NB18 CCAAACCCAACAACCTAGATAGGC GCCTATCTAGGTTGTTGGGTTTGG NB19 GTTCCTCGTGCAGTGTCAAGAGAT ATCTCTTGACACTGCACGAGGAAC NB20 TTGCGTCCTGTTACGAGAACTCAT ATGAGTTCTCGTAACAGGACGCAA NB21 GAGCCTCTCATTGTCCGTTCTCTA TAGAGAACGGACAATGAGAGGCTC NB22 ACCACTGCCATGTATCAAAGTACG CGTACTTTGATACATGGCAGTGGT NB23 CTTACTACCCAGTGAACCTCCTCG CGAGGAGGTTCACTGGGTAGTAAG NB24 GCATAGTTCTGCATGATGGGTTAG CTAACCCATCATGCAGAACTATGC