- Materials
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- 3.5 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)
- 1.5 ml Eppendorf DNA LoBind tubes
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind 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 3.5 μg genomic DNA into a DNA LoBind tube
- Adjust the volume to 16 μl or less with nuclease-free water
- Mix thoroughly by inversion avoiding unwanted shearing
- Spin down briefly in a microfuge
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Fragment DNA to ~1-2 kb as follows:
Mix the reagents in the order given below:
Reagent Volume DNA (1-16 µl) x µl 10x Fragmentase Reaction Buffer v2 2 µl Nuclease-free water 16 - x µl dsDNA Fragmentase 2 µl Total 20 µl -
Vortex for 3 sec.
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Incubate the reaction for 10 minutes at 16° C.
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Resuspend the AMPure XP beads by vortexing.
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Add 36 µl of the AMPure XP beads to the reaction and mix gently by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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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 48 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 48 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of fragmented and repaired DNA using a Qubit fluorometer