- 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™ cat # A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- 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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- Microfuge
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Ice bucket with ice
- Multichannel pipette and tips
- Magnetic rack
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:
Thaw the reagents at room temperature.
Spin down the reagent tubes for 5 seconds.
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
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Spin down the Barcode Adapter (BCA), pipette mix and place on ice.
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Add the reagents in the order given below, into fresh 0.2 ml PCR tubes or 96-well plate:
Between each addition, pipette mix 10-20 times.
Reagent Volume End-prepped DNA 15 µl Barcode Adapter 10 µl Blunt/TA Ligase Master Mix 25 µl Total 50 µl -
Mix by pipetting and briefly spin down.
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Incubate the samples for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 20 µl of resuspended AMPure XP beads to each sample for a 0.4X clean and mix by pipetting up and down ten times.
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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 sample, with some excess.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Keep the samples 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.
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Repeat the previous step.
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Place the samples back on the magnet. Pipette off any residual 80% ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the samples 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 and retain the eluate once it is clear and colourless. Transfer each eluted sample to a fresh 0.2 ml PCR tube or plate.
- Dispose of the pelleted beads.
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Quantify 1 µl of the adapter ligated DNA using a Qubit fluorometer.