- Materials
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- Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing more than 12 samples
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Equipment
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- Microfuge
- Ice bucket with ice
- Hula mixer (gentle rotator mixer)
- Magnetic rack
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Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:
Reagent Volume End-prepped DNA 22.5 µl Native Barcode 2.5 µl Blunt/TA Ligase Master Mix 25 µl Total 50 µl -
Mix gently by flicking the tube, and spin down.
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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 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% 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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Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry.
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Remove the tube from the magnetic rack and resuspend the pellet in 26 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 26 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.
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Pool the barcoded samples at the desired ratio to a final volume of 65 μl in a 1.5 ml Eppendorf DNA LoBind tube. Aim for as high a concentration as possible which does not exceed 200 fmol total. If the total volume is >65 μl, perform a 2.5x AMPure clean up and elute in 65 μl of nuclease free water.