- Materials
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- Up to 24 pools of PCR barcoded sample pools (with up to 96 samples in each pool), each in 12.5 µl
- AMPure XP Beads (AXP)
- Consumables
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- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Freshly prepared 80% ethanol in nuclease-free water
- 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
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 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
- Multichannel pipette and tips
- Thermal cycler
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- Ice bucket with ice
- Magnetic rack
- Vortex mixer
- Hula mixer (rotator mixer)
- Qubit fluorometer (or equivalent)
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Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing. Keep the beads at room temperature until use.
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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 clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), prepare 200 fmol (130 ng for 1 kb amplicons) of each PCR barcoded sample pool.
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Make up each PCR barcoded sample pool to 12.5 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 PCR barcoded sample pool 12.5 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 PCR barcoded sample pool 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 sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per PCR barcoded sample pool, with some excess.
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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 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 end-prepped PCR barcoded sample pool using a Qubit fluorometer.