- Materials
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- 1–5 ng high molecular weight genomic DNA
- Rapid PCR Barcoding Kit (SQK-RPB004)
- Flow Cell Priming Kit (EXP-FLP002)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- P1000 pipette tips
- P200 pipette tips
- P100 pipette tips
- P20 pipette tips
- P10 pipette tips
- P2 pipette tips
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Freshly prepared 70% ethanol in nuclease-free water
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
- Equipment
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- Microfuge
- P1000 pipette
- P200 pipette
- P100 pipette
- P20 pipette
- P10 pipette
- P2 pipette
- Timer
- Thermal cycler
- 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 1-5 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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Rapid PCR Barcoding Kit (SQK-RPB004) contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Fragmentation Mix FRM Brown 3 30 Rapid Adapter RAP Green 1 10 Sequencing Buffer SBQ Red 1 300 Loading Beads LB Pink 1 360 Rapid Barcode Primer 1-12 RLB 01-12 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