- Materials
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- 1 µg (or 100-200 fmol) gDNA
- 1.5-3 µg (or 150-300 fmol) high molecular weight genomic DNA if using R10.3 flow cells
- OR 100+ ng high molecular weight genomic DNA if performing DNA fragmentation
- Ligation Sequencing Kit XL (SQK-LSK109-XL)
- Flow Cell Priming Kit XL (EXP-FLP002-XL)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L).
Alternatively, you can use the NEBNext® products below:
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- OR 0.2 ml thin-walled PCR tubes
- 15 or 50 ml Falcon tubes
- Equipment
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- Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
- OR magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Side Magnet (Thermo Fisher, #12331D)
- Microfuge
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Vortex mixer
- Thermal cycler
- Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, 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
- Pipetting troughs
- 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 1 µg (or 100-200 fmol) gDNA.
Although 1 µg (or 100-200 fmol) gDNA is recommended, users can start with lower input quantities (down to 100 ng) if performing DNA fragmentation to increase the number of DNA molecules in the sample, or if amplifying the sample by PCR.
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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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NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.
Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.
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Multichannel pipettes
For scaling up library prep using the Ligation Sequencing Kit XL, customers will need multichannel pipettes and appropriate pipette tips. Although the choice of brand is left to the user's discretion, our R&D team can recommend Rainin Pipet-Lite LTS L200-XLS+ (20–200 μl) and Pipet-Lit LTS L20-XLS+ (2–20 μl) pipettes and Rainin LTS pipette tips.
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Ligation Sequencing Kit XL (SQK-LSK109-XL) contents
Name Acronym Cap colour No. of tubes Full volume (μl) DNA CS DCS Yellow 1 52 Adapter Mix AMX Green 4 60 Ligation Buffer LNB Clear 4 300 L Fragment Buffer LFB White cap, orange stripe on label 4 6,000 S Fragment Buffer SFB Grey 4 6,000 Sequencing Buffer SQB Red 4 900 Elution Buffer EB Black 1 1,200 Loading Beads LB Pink 4 612 -
Flow Cell Priming Kit XL (EXP-FLP002-XL) contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Flush Buffer FB Blue 4 15,400 Flush Tether FLT Purple 4 400