- Materials
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- cDNA in 20 μl nuclease-free water
- 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™, A63881)
- Equipment
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- Thermal cycler
- Magnetic rack
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Perform end repair and dA-tailing of the cDNA sample as follows:
Mix the following reagents in a 0.2 ml PCR tube:
Reagent Volume cDNA sample 20 µl Nuclease-free water 30 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Total 60 µl -
Mix gently 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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Resuspend the AMPure XP beads by vortexing.
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Transfer the sample to a 1.5 ml DNA LoBind Eppendorf tube.
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Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
End-prep cleanup demo
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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.
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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 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 30 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 30 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.