- Materials
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- 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded
- AMPure XP Beads (AXP)
- 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)
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Ice bucket with ice
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- 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 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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Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing. Keep the beads at room temperature.
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In clean 0.2 ml thin-walled PCR tubes, aliquot 200 fmol (130 ng for 1 kb amplicons) of DNA per sample.
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Make up each sample to 12.5 µl using nuclease-free water. Mix gently by pipetting and spin down.
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Combine the following components per sample:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume Ultra II End-prep reaction buffer 1.75 µl Ultra II End-prep enzyme mix 0.75 µl Total 2.5 µl -
Mix well by pipetting and spin down in a centrifuge.
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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 the reaction to a clean 1.5 ml Eppendorf DNA LoBind tube per sample.
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Resuspend the AMPure XP Beads (AXP) by vortexing.
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Add 15 µl of resuspended AMPure XP Beads (AXP) to each 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 samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
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Keep the tubes on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellets. 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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Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
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Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and 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 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube per sample.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.