- Materials
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- Barcode Adapter (BCA)
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 10 mM Tris-HCl pH 8.5
- 0.2 ml 96-well PCR plate
- Equipment
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- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
- Ice bucket with ice
- Multichannel pipette and tips
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
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Add the reagents to a fresh 96-well plate, in the order given below:
Between each addition, pipette mix 10-20 times.
Reagent Volume End-prepped DNA 30 µl Barcode Adapter 20 µl Blunt/TA Ligase Master Mix 50 µl Total 100 µl -
Ensure the components are thoroughly mixed by pipetting.
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Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 40 µl of resuspended AMPure XP beads to each sample and mix by pipetting up and down ten times.
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Allow DNA to bind to beads for 5 minutes at room temperature.
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Prepare sufficient fresh 70% ethanol in nuclease-free water.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Keep the tube on the magnet and wash the beads with 180 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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Cover the plate with adhesive film and leave plate on magnet for 2 minutes to allow residual liquid to collect at the bottom. Remove the adhesive film, return the plate to the magnet and aspirate residual wash solution.
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Briefly incubate the plate on a thermal cycler at 37° C with the lid open and the plate wells unsealed.
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Remove the plate from the magnet and resuspend pellet in 25 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.
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Remove eluate once it is clear and colourless. Transfer each eluted sample to a new 96-well PCR plate.
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Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.
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Dilute the library to a concentration of 10 ng/µl with nuclease-free water or 10 mM Tris-HCl pH 8.5.