- Materials
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- 200 fmols of UMI tagged amplicons
- Consumables
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- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 0.2 ml PCR tubes
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Equipment
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- Microfuge
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack suitable for 0.2 ml PCR tubes
- Qubit fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Vortex mixer
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Determine the volume of the cleaned-up PCR reaction that yields 200 fmols of UMI tagged amplicons.
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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 perfomance, NEB recommend the following:
Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they 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 Ultra II End Prep 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 30 seconds to solubilise any precipitate.
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In a 0.2 ml thin-walled PCR tube, combine the following:
Reagent Volume UMI tagged amplicons 200 fmols Nuclease-free water Up to 50 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Total 60 µl -
Mix gently by flicking the tube and spin down.
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Using a thermal cycler, incubate at 20°C for 10 minutes and 65°C for 10 minutes.
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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 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 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet until the eluate is clear and colourless. Keep the plate on the magnetic rack, 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 70% ethanol without disturbing the pellet. Wait for the beads to migrate towards the magnet and form a 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 63 µl nuclease-free water. Spin down and incubate for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear.
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Remove and retain 63 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.