- Materials
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- Native Barcoding Expansion 96 (EXP-NBD196)
- Short Fragment Buffer (SFB)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- NEB Blunt/TA Ligase Master Mix (M0367)
- Equipment
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- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Ice bucket with ice
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Microfuge
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Thaw Native Barcodes at room temperature. Use one barcode per sample.
- Spin down barcodes before opening tubes/piercing plates.
- Pipette mix the entire content of each barcode 10 times before use.
- Once thawed, keep the barcodes on ice.
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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 Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.
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In a clean 96-well plate (Native barcode ligation plate), add the following reagents in the following order per well:
Between each addition, pipette mix 10-20 times.
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, seal the Native barcode ligation plate and spin down briefly.
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Using a thermal cycler, incubate the Native barcode ligation plate at 20°C for 20 mins and at 65°C for 10 mins.
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Pool all the barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube, checking the base of the plate to ensure all the liquid has been pooled.
Recovery aim ~ 10 µl per sample:
24 samples 48 samples 96 samples Total volume ~ 240 µl ~ 480 µl ~ 960 µl -
Resuspend the AMPure XP beads by vortexing.
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Add a 0.4X volume of AMPure XP beads to the pooled reaction and mix thoroughly by pipetting:
Volume per sample For X24 samples For X48 samples For X96 samples Volume of AMPure XP beads 4 µl 96 µl 192 µl 384 µl -
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 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 magnetic rack 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 magnetic rack. Pipette off any residual ethanol. Allow the pellet 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 magnetic rack 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.
Dispose of the pelleted beads
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Quantify 1 µl of eluted sample using a Qubit fluorometer.