- Materials
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- 1–5 ng high molecular weight genomic DNA
- Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Ice bucket with ice
- Microfuge
- Timer
- Thermal cycler
- Magnetic rack
- Hula mixer (gentle rotator mixer)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
- Qubit fluorometer (or equivalent for QC check)
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
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For this protocol, you will need 1-5 ng high molecular weight genomic DNA.
Note: Your input DNA must be at least 4 kb in length to ensure correct tagmentation and PCR amplification.
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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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Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24) contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Fragmentation Mix FRM Brown 1 160 Rapid Adapter RA Green 1 15 Adapter Buffer ADB Clear 1 100 AMPure XP Beads AXP Amber 3 1,200 Elution Buffer EB Black 2 500 EDTA EDTA Blue 1 700 Sequencing Buffer SB Red 1 700 Library Beads LIB Pink 1 600 Library Solution LS White cap, pink label 1 600 Flow Cell Flush FCF Clear 1 8,000 Flow Cell Tether FCT Purple 1 200 Rapid Barcode Primer 01-24 RLB01-24 Clear 24 (one per barcode) 15 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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To maximise the use of the Rapid Barcoding Kits, the Rapid Adapter Auxiliary V14 (EXP-RAA114) and the Sequencing Auxiliary Vials V14 (EXP-AUX003) expansion packs are available.
These expansions provide additional library preparation and flow cell priming reagents to allow users to utilise any unused barcodes for those running in smaller subsets.
Both expansion packs used together will provide enough reagents for 6 library preparations.
Rapid Adapter Auxiliary V14 (EXP-RAA114) contents:
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Rapid Adapter RA 1 Green 15 Adapter Buffer ADB 1 Clear 100 Sequencing Auxiliary Vials V14 (EXP-AUX003) contents:
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Elution Buffer EB Black 2 500 Sequencing Buffer SB Red 2 700 Library Solution LIS White cap, pink label 2 600 Library Beads LIB Pink 2 600 Flow Cell Flush FCF Light blue label 2 8,000 Flow Cell Tether FCT Purple 2 200 -
Rapid barcode primers
Component Sequence RLB01 AAGAAAGTTGTCGGTGTCTTTGTG RLB02 TCGATTCCGTTTGTAGTCGTCTGT RLB03 GAGTCTTGTGTCCCAGTTACCAGG RLB04 TTCGGATTCTATCGTGTTTCCCTA RLB05 CTTGTCCAGGGTTTGTGTAACCTT RLB06 TTCTCGCAAAGGCAGAAAGTAGTC RLB07 GTGTTACCGTGGGAATGAATCCTT RLB08 TTCAGGGAACAAACCAAGTTACGT RLB09 AACTAGGCACAGCGAGTCTTGGTT RLB10 AAGCGTTGAAACCTTTGTCCTCTC RLB11 GTTTCATCTATCGGAGGGAATGGA RLB12 GTTGAGTTACAAAGCACCGATCAG RLB13 AGAACGACTTCCATACTCGTGTGA RLB14 AACGAGTCTCTTGGGACCCATAGA RLB15 AGGTCTACCTCGCTAACACCACTG RLB16 CGTCAACTGACAGTGGTTCGTACT RLB17 ACCCTCCAGGAAAGTACCTCTGAT RLB18 CCAAACCCAACAACCTAGATAGGC RLB19 GTTCCTCGTGCAGTGTCAAGAGAT RLB20 TTGCGTCCTGTTACGAGAACTCAT RLB21 GAGCCTCTCATTGTCCGTTCTCTA RLB22 ACCACTGCCATGTATCAAAGTACG RLB23 CTTACTACCCAGTGAACCTCCTCG RLB24 GCATAGTTCTGCATGATGGGTTAG