- <1 µg (or <100–200 fmol) high molecular weight genomic DNA
- Ligation Sequencing Kit (SQK-LSK110)
- PCR Expansion (EXP-PCA001)
- 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)
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- 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
- 10 mM Tris-HCl pH 8.5
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Vortex mixer
- Thermal cycler
- 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
- Optional equipment
- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
For this protocol, you will need <100–200 fmol high molecular weight genomic 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.
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.
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.
Ligation Sequencing Kit (SQK-LSK110) contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) DNA CS DCS Yellow 1 35 Adapter Mix F AMX-F Green 1 40 Ligation Buffer LNB Clear 1 200 L Fragment Buffer LFB White cap, orange stripe on label 2 1,800 S Fragment Buffer SFB Grey 2 1,800 Sequencing Buffer II SBII Red 1 500 Elution Buffer EB Black 1 200 Loading Beads II LBII Pink 1 360 Loading Solution LS White cap, pink sticker on label 1 360 Flush Buffer FB Blue 6 1,170 Flush Tether FLT Purple 1 200
PCR Expansion (EXP-PCA001) contents
Name Acronym Cap colour No. of vials Fill volume per vial (μl) PCR Primers PRM White 4 20 PCR Adapters PCA Blue 4 140