- Materials
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- 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
- Phosphatase (PHOS)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler
- P100 pipette and tips
- P10 pipette and tips
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This step reduces background reads by removing 5’ phosphates from non-target DNA ends.
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Prepare the DNA in nuclease-free water
- Transfer 1-10 μg (with 5 μg recommended) genomic DNA into a 0.2 ml thin-walled PCR tubes
- Adjust to 24 µl with nuclease-free water
- Mix thoroughly by flicking the tube avoiding unwanted shearing
- Spin down briefly in a microfuge
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Mix the Phosphatase (PHOS) in the tube by pipetting up and down. Ensure that it is at room temperature before use.
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Assemble the following components in a clean 0.2 ml thin-walled PCR tube:
Reagent Volume Reaction Buffer (RB) 3 µl HMW genomic DNA (at ≥ 210 ng/µl)* 24 µl Total 27 µl - Note: For an initial test, we recommend 5 µg genomic DNA input. Preparing input DNA step yields ~100-2000x target coverage. Target coverage scales linearly with input amount, so the input amount may be reduced accordingly if lower throughput is acceptable.
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Ensure the components are thoroughly mixed by pipetting, and spin down.
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Add 3 µl of PHOS to the tube.
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Mix gently by flicking the tube, and spin down.
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Using a thermal cycler, incubate at 37ºC for 10 minutes, 80ºC for 2 minutes then hold at 20ºC (room temperature).