- Materials
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- 100-200 fmol first-round PCR product in 24 μl (with tailed primers, see full protocol for further details)
- PCR Barcoding Expansion 1-96 (EXP-PBC096)
- Ligation Sequencing Kit (SQK-LSK110)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L).
Alternatively, you can use the NEBNext® products below:
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- 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
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- 0.2 ml 96-well PCR plate
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- 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
- Timer
- Multichannel pipette and tips
- 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 100-200 fmol first-round PCR product with tailed primers in 24 μl 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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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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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 Barcoding Expansion Pack 1-96 (EXP-PBC096)
Name Acronym Cap colour No. of vials/plates Fill volume per well (µl) PCR Barcode Primer Mix plate BC01-96 White 1 plate 24 Barcode Adapter plate BCA Blue 1 plate 240 -
Layout of barcodes in the 96 tube plate
The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.