- Materials
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- Fragmented DNA in 20 µl
- Consumables
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- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 80% ethanol in nuclease-free water
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
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In a 0.2 ml thin-walled PCR tube, mix the following:
Between each addition, pipette mix 10-20 times.
Reagent Volume Fragmented DNA 20 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Nuclease-free water 30 µl Total 60 µl -
Ensure the components are thoroughly mixed by pipetting, and spin down.
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Using a thermal cycler, incubate at 20°C for 30 minutes and 65°C for 30 mins.
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Resuspend the AMPure XP beads by vortexing.
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Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by flicking the tube.
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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 80% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, 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 31 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, for at least 1 minute.
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Remove and retain 31 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.