- Materials
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- Native Barcodes (NB01-24)
- Native Barcodes (NB01-NB96)
- Short Fragment Buffer (SFB)
- Consumables
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- Freshly prepared 80% ethanol in nuclease-free water
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Equipment
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- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- Thermal cycler
- Microfuge
- Centrifuge capable of taking 96-well plates
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Hula mixer (gentle rotator mixer)
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
- Optional equipment
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- PCR-Cooler (Eppendorf)
- Stepper pipette and tips
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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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Using a stepper pipette, or a multichannel pipette, make up the NB Ligation Plate in a clean 96-well plate. Add the reagents in the following order per well:
Reagent Volume per well for up to x24 samples Volume per well for x25—x96 samples Nuclease-free water 6 µl 3 µl End-prepped DNA
Note: Transfer to the corresponding well1.5 µl 0.75 µl Native barcode
Note: Transfer to the corresponding well2.5 µl 1.25 µl Blunt/TA Ligation Master Mix 10 µl 5 µl Total 20 µl 10 µl Depending on the number of samples, aliquot to each well of the columns:
Plate location x24 samples x48 samples x96 samples Columns 1-3 1-6 1-12 -
Mix the contents thoroughly by pipetting.
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Seal the plate 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 samples.
- For up to x24 samples, we expect ~20 µl per sample.
- For x25—x96 samples, we expect ~10 µl per sample.
x24 samples x48 samples x96 samples Total volume ~480 µl ~480 µl ~960 µl -
Take forward 480 µl of the pooled barcoded samples.
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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 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.
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Quantify 1 µl of eluted sample using a Qubit fluorometer - recovery aim 2 ng/μl.