- Materials
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- 100 ng fragmented DNA in 50 µl
- Consumables
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- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Equipment
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- Thermal cycler
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Ice bucket with ice
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Prepare the NEBNext Ultra II End Repair/dA-Tailing Module reagents according to the manufacturer’s instructions, and place on ice.
For optimal performance, NEB recommend the following:
- Thaw all reagents on ice.
- Flick and/or invert reagent tube to ensure they are well mixed.
- Always spin down tubes before opening for the first time each day.
- The Ultra II End prep buffer and FFPE DNA Repair buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.
- The FFPE DNA repair buffer may have a yellow tinge and is fine to use if yellow.
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Mix the following reagents in a 0.2 ml thin-walled PCR tube:
Reagent Volume 100 ng fragmented DNA 50 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Total 60 µl -
Mix well by gently pipetting the entire volume within the tube up and down 10 times.
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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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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 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 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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Repeat the previous step.
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Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry.
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Remove the tube from the magnetic rack and resuspend pellet in 16 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 16 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.