- Materials
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- Native Barcoding Expansion 96 (EXP-NBD196)
- Short Fragment Buffer (SFB)
- Consumables
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- Freshly prepared 80% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- Equipment
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- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Vortex mixer
- Ice bucket with ice
- Microfuge
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Optional equipment
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- 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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Thaw the native barcodes at room temperature. Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.
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Thaw the tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
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Select a unique barcode for every sample to be run.
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In a new 96-well plate, add the reagents in the order given below per well:
Mix well by pipetting and spin down in a centrifuge.
Reagent Volume Nuclease-free water 3 µl End-prepped DNA 0.75 µl Native Barcode 1.25 µl Blunt/TA Ligase Master Mix 5 µl Total 10 µl -
Mix contents thoroughly by pipetting and spin down briefly.
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Using a thermal cycler, incubate at 20°C for 20 mins and at 65°C for 10 mins.
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Pool the barcoded library together and carry forward 480 µl of the library.
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Resuspend the AMPure XP beads by vortexing.
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Add 192 µl of resuspended AMPure XP beads to the 480 µl of pooled reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 10 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 sample and pellet the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
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Wash the beads by adding 700 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Keep the tube on the magnet until the eluate is clear and colourless. Remove the supernatant using a pipette and discard.
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Repeat the previous step.
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Keep the tube on the magnet and wash the beads with 100 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the tube from the magnetic rack and resuspend the pellet in 35 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 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of eluted sample using a Qubit fluorometer - recovery aim 2 ng/μl.