- Materials
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- 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded
- AMPure XP Beads (AXP)
- DNA Control Sample (DCS)
- Consumables
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- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- OR 0.2 ml thin-walled PCR tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
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- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Magnetic rack
- Multichannel pipette and tips
- Vortex mixer
- Hula mixer (rotator mixer)
- Thermal cycler
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
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Thaw the AMPure XP Beads (AXP) and DNA Control Sample (DCS) at room temperature and mix by vortexing. Keep the beads at room temperature and store the DNA Control Sample (DCS) on ice.
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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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Dilute your DNA Control Sample (DCS) by adding 105 µl Elution Buffer (EB) directly to one DCS tube. Mix gently by pipetting and spin down.
One tube of diluted DNA Control Sample (DCS) is enough for 140 samples. Excess can be stored at -20°C in the freezer.
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In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), aliquot 200 fmol (130 ng for 1 kb amplicons) of DNA per sample.
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Make up each sample to 11.5 µl using nuclease-free water. Mix gently by pipetting and spin down.
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Combine the following components per tube/well:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume 200 fmol (130 ng for 1 kb amplicons) amplicon DNA 11.5 µl Diluted DNA Control Sample (DCS) 1 µl Ultra II End-prep Reaction Buffer 1.75 µl Ultra II End-prep Enzyme Mix 0.75 µl Total 15 µl -
Ensure the components are thoroughly mixed 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 each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
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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 80% 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 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.
If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
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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.
- Dispose of the pelleted beads
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.