- Materials
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- 50 ng high molecular weight plasmid DNA per sample
- Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) OR Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
- Consumables
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- 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
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
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- MinION or GridION device
- Ice bucket with ice
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Timer
- Thermal cycler or heat blocks
- 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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- Standard gel electrophoresis equipment
- Agilent Bioanalyzer (or equivalent)
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For this protocol, you will need 50 ng high molecular weight plasmid DNA per sample.
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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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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.
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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 sequences
Component Sequence RB01 AAGAAAGTTGTCGGTGTCTTTGTG RB02 TCGATTCCGTTTGTAGTCGTCTGT RB03 GAGTCTTGTGTCCCAGTTACCAGG RB04 TTCGGATTCTATCGTGTTTCCCTA RB05 CTTGTCCAGGGTTTGTGTAACCTT RB06 TTCTCGCAAAGGCAGAAAGTAGTC RB07 GTGTTACCGTGGGAATGAATCCTT RB08 TTCAGGGAACAAACCAAGTTACGT RB09 AACTAGGCACAGCGAGTCTTGGTT RB10 AAGCGTTGAAACCTTTGTCCTCTC RB11 GTTTCATCTATCGGAGGGAATGGA RB12 CAGGTAGAAAGAAGCAGAATCGGA RB13 AGAACGACTTCCATACTCGTGTGA RB14 AACGAGTCTCTTGGGACCCATAGA RB15 AGGTCTACCTCGCTAACACCACTG RB16 CGTCAACTGACAGTGGTTCGTACT RB17 ACCCTCCAGGAAAGTACCTCTGAT RB18 CCAAACCCAACAACCTAGATAGGC RB19 GTTCCTCGTGCAGTGTCAAGAGAT RB20 TTGCGTCCTGTTACGAGAACTCAT RB21 GAGCCTCTCATTGTCCGTTCTCTA RB22 ACCACTGCCATGTATCAAAGTACG RB23 CTTACTACCCAGTGAACCTCCTCG RB24 GCATAGTTCTGCATGATGGGTTAG RB25 GTAAGTTGGGTATGCAACGCAATG RB26 CATACAGCGACTACGCATTCTCAT RB27 CGACGGTTAGATTCACCTCTTACA RB28 TGAAACCTAAGAAGGCACCGTATC RB29 CTAGACACCTTGGGTTGACAGACC RB30 TCAGTGAGGATCTACTTCGACCCA RB31 TGCGTACAGCAATCAGTTACATTG RB32 CCAGTAGAAGTCCGACAACGTCAT RB33 CAGACTTGGTACGGTTGGGTAACT RB34 GGACGAAGAACTCAAGTCAAAGGC RB35 CTACTTACGAAGCTGAGGGACTGC RB36 ATGTCCCAGTTAGAGGAGGAAACA RB37 GCTTGCGATTGATGCTTAGTATCA RB38 ACCACAGGAGGACGATACAGAGAA RB39 CCACAGTGTCAACTAGAGCCTCTC RB40 TAGTTTGGATGACCAAGGATAGCC RB41 GGAGTTCGTCCAGAGAAGTACACG RB42 CTACGTGTAAGGCATACCTGCCAG RB43 CTTTCGTTGTTGACTCGACGGTAG RB44 AGTAGAAAGGGTTCCTTCCCACTC RB45 GATCCAACAGAGATGCCTTCAGTG RB46 GCTGTGTTCCACTTCATTCTCCTG RB47 GTGCAACTTTCCCACAGGTAGTTC RB48 CATCTGGAACGTGGTACACCTGTA RB49 ACTGGTGCAGCTTTGAACATCTAG RB50 ATGGACTTTGGTAACTTCCTGCGT RB51 GTTGAATGAGCCTACTGGGTCCTC RB52 TGAGAGACAAGATTGTTCGTGGAC RB53 AGATTCAGACCGTCTCATGCAAAG RB54 CAAGAGCTTTGACTAAGGAGCATG RB55 TGGAAGATGAGACCCTGATCTACG RB56 TCACTACTCAACAGGTGGCATGAA RB57 GCTAGGTCAATCTCCTTCGGAAGT RB58 CAGGTTACTCCTCCGTGAGTCTGA RB59 TCAATCAAGAAGGGAAAGCAAGGT RB60 CATGTTCAACCAAGGCTTCTATGG RB61 AGAGGGTACTATGTGCCTCAGCAC RB62 CACCCACACTTACTTCAGGACGTA RB63 TTCTGAAGTTCCTGGGTCTTGAAC RB64 GACAGACACCGTTCATCGACTTTC RB65 TTCTCAGTCTTCCTCCAGACAAGG RB66 CCGATCCTTGTGGCTTCTAACTTC RB67 GTTTGTCATACTCGTGTGCTCACC RB68 GAATCTAAGCAAACACGAAGGTGG RB69 TACAGTCCGAGCCTCATGTGATCT RB70 ACCGAGATCCTACGAATGGAGTGT RB71 CCTGGGAGCATCAGGTAGTAACAG RB72 TAGCTGACTGTCTTCCATACCGAC RB73 AAGAAACAGGATGACAGAACCCTC RB74 TACAAGCATCCCAACACTTCCACT RB75 GACCATTGTGATGAACCCTGTTGT RB76 ATGCTTGTTACATCAACCCTGGAC RB77 CGACCTGTTTCTCAGGGATACAAC RB78 AACAACCGAACCTTTGAATCAGAA RB79 TCTCGGAGATAGTTCTCACTGCTG RB80 CGGATGAACATAGGATAGCGATTC RB81 CCTCATCTTGTGAAGTTGTTTCGG RB82 ACGGTATGTCGAGTTCCAGGACTA RB83 TGGCTTGATCTAGGTAAGGTCGAA RB84 GTAGTGGACCTAGAACCTGTGCCA RB85 AACGGAGGAGTTAGTTGGATGATC RB86 AGGTGATCCCAACAAGCGTAAGTA RB87 TACATGCTCCTGTTGTTAGGGAGG RB88 TCTTCTACTACCGATCCGAAGCAG RB89 ACAGCATCAATGTTTGGCTAGTTG RB90 GATGTAGAGGGTACGGTTTGAGGC RB91 GGCTCCATAGGAACTCACGCTACT RB92 TTGTGAGTGGAAAGATACAGGACC RB93 AGTTTCCATCACTTCAGACTTGGG RB94 GATTGTCCTCAAACTGCCACCTAC RB95 CCTGTCTGGAAGAAGAATGGACTT RB96 CTGAACGGTCATAGAGTCCACCAT