- Materials
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- AMPure XP Beads (AXP)
- Consumables
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- NEBNext® Ultra II End Prep Enzyme Mix from NEBNext® Ultra II End Repair Module (NEB, E7546)
- NEBNext® Ultra II End Prep Reaction Buffer from NEBNext® Ultra II End Repair Module (NEB, E7546)
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- 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
- 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 in accordance with manufacturer's instructions, and place on ice:
For optimal performance, NEB recommend the following:
Thaw all reagents on ice.
Ensure the reagents are well mixed.
Note: Do not vortex the Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The NEBNext Ultra II End Prep Reaction Buffer may contain a white precipitate. If this occurs, allow the mixture(s) to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.
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Prepare the PCR amplified DNA in nuclease-free water
- Transfer 1 ug genomic DNA into a 0.2 ml thin-walled PCR tube.
- Adjust the volume to 50 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 50 µl Ultra II End-prep Reaction Buffer 7 µl Ultra II End-prep Enzyme Mix 3 µl Total 60 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning 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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Resuspend the AMPure XP Beads (AXP) 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 the AMPure XP Beads (AXP) 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 the pellet in 61 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 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of eluted sample using a Qubit fluorometer.