- Materials
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- <1 µg of each DNA sample to be barcoded in 45 µl
- Consumables
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- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- Freshly prepared 70% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler
- Ice bucket with ice
- Microfuge
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Prepare the DNA in nuclease-free water.
- Transfer <1 μg DNA of each sample into a fresh 0.2 ml PCR tube or plate
- Adjust the volume to 45 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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Using a separate 0.2 ml thin-walled PCR tube for each DNA sample to be barcoded, set up the end-repair / dA-tailing reactions as follows:
Between each addition, pipette mix 10-20 times.
Reagent Volume <1 µg DNA 45 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Nuclease-free water 5 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 5 minutes and 65°C for 5 minutes.
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Transfer each sample to a separate 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the AMPure XP beads by vortexing.
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Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare sufficient fresh 70% ethanol in nuclease-free water.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Keep the tube on the magnet and wash the beads with 180 µl of freshly-prepared 70% 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 tubes 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 tubes from the magnetic rack and resuspend the 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.
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Remove eluate once it is clear and colourless. Transfer each eluted sample to a new 0.2 ml thin-walled PCR tube.
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Quantify 1 µl of end-prepped DNA using a Qubit fluorometer - recovery aim >700 ng.