- Materials
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- 50 pg high molecular weight genomic DNA
- Ligation Sequencing Kit V14 (SQK-LSK114)
- REPLI-g® Single Cell Kit (QIAGEN, cat # 150343)
- Consumables
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- MinION and GridION Flow Cell
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7672S or E7672L)
- Salt-T4® DNA Ligase (NEB, M0467)
- Covaris g-TUBE
- 2 ml Eppendorf DNA LoBind tubes
- 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
- T7 Endonuclease I (NEB, cat # M0302)
- TE buffer: 10 mM Tris (pH 8.0), 0.1 mM EDTA
- PEG 8000, 50% w/v (Rigaku Reagents, 25322-68-3)
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- 5 M NaCl (Sigma, 71386)
- 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
- Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- Equipment
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- MinION or GridION device
- MinION and GridION Flow Cell Light Shield
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Heating block at 37°C capable of taking 1.5 ml tubes
- Thermal cycler
- Ice bucket with ice
- Timer
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
- Optional equipment
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- Standard gel electrophoresis equipment
- Agilent Bioanalyzer (or equivalent)
- Eppendorf 5424 centrifuge (or equivalent)
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For this protocol, you will need 50 pg high molecular weight genomic DNA.
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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 v2 for Oxford Nanopore Technologies® Ligation Sequencing
We recommend buying the NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7672S or E7672L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.
The previous version, NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L) is compatible, but the recommended v2 module offers more efficient dA-tailing and ligation, a result of the FFPEv2 DNA Repair Buffer and Salt-T4 DNA Ligase, respectively.
A marked cost saving per sample preparation is also realised when using the v2 module.Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix is not required and purchasing the required reagents separately is more cost effective.
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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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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 -
Ligation Sequencing Kit V14 (SQK-LSK114) contents
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format.
Single-use tubes format:
Bottle format:
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.
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.