- Materials
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- 3 µg high molecular weight human genomic DNA
- Consumables
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- NEBNext dsDNA Fragmentase (M0348L)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 80% ethanol in nuclease-free water
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Ice bucket with ice
- Microfuge
- Thermal cycler
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Eppendorf 5424 centrifuge (or equivalent)
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Prepare the DNA in nuclease-free water.
- Transfer 3 μg genomic DNA into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to 16 μl with nuclease-free water
- Mix thoroughly by flicking the tube to avoid unwanted shearing
- Spin down briefly in a microfuge
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In a 0.2 ml thin-walled PCR tube, mix the following:
Reagent Volume DNA 16 µl 10x Fragmentase Reaction Buffer v2 2 µl Total 18 µl -
Vortex the tube for 3 seconds, and spin down.
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Add 2 μl dsDNA Fragmentase to the tube.
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Incubate the reaction for 28 minutes at 37°C.
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Stop the fragmentation reaction by adding 5 μl of 0.5 M EDTA to the tube.
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Vortex the tube for 3 seconds, and spin down.
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Resuspend the AMPure XP beads by vortexing.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Add 15 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Spin down the sample and pellet the beads on a magnet for 5 mins.
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Remove and retain the supernatant in a new 1.5 ml Eppendorf DNA LoBind tube.
Note: Do not discard the supernatant.
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Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Prepare 500 μl of fresh 80% ethanol in nuclease-free water.
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Spin down the sample and pellet the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Run a 1 μl aliquot on an Agilent Bioanalyzer to determine fragment length.
The fragment length distribution is expected to be similar to the trace below: