- Materials
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- 100 ng high molecular weight genomic DNA
- PCR Barcoding Kit (SQK-PBK004)
- Flow Cell Priming Kit (EXP-FLP002)
- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- Covaris g-TUBE
- 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 Hot Start Taq 2X Master Mix (NEB, M0533S)
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
- (optional) Exonuclease I (NEB, M0293)
- Equipment
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- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Ice bucket with ice
- Timer
- 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
- Optional equipment
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- Standard gel electrophoresis equipment
- 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 ng 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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PCR Barcoding Kit contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Barcode Adapter BCA Blue 3 260 Rapid Adapter RAP Green 1 10 Loading Beads LB Pink 1 360 Sequencing Buffer SQB Red 1 300 Barcode Primer 01-12 BP01-BP12 Clear 12 10 -
Flow Cell Priming Kit contents (EXP-FLP002)
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Flush Buffer FB Blue 6 1,170 Flush Tether FLT Purple 1 200 -
The RAP Top-Up Kit (EXP-RAP001) is available to provide enough reagents for another six reactions depending on how the barcodes are used.
This kit contains reagents to be used with any remaining barcodes to load another six sequencing libraries.
Reagent Acronym Cap colour No. of vials Fill volume per vial (µl) Rapid Adapter RAP Green 1 10 Sequencing Tether SQT Purple 1 10 Loading Beads LB Pink 1 360 Sequencing Buffer SQB Red 1 300