- Materials
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- Native Barcodes (NB01-24) OR Native Barcodes (NB01-NB96)
- AMPure XP Beads (AXP)
- EDTA (EDTA)
- 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)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- Equipment
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- Magnetic rack suitable for 96-well plates
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- 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 kit components at room temperature, then spin down briefly using a microfuge and mix as indicated by the table below. Then place on ice:
Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix by pipetting or vortexing 4. Place on ice EDTA (EDTA) ✓ ✓ Vortexing ✓ Native Barcodes (NB01-24) or (NB01-96) Not frozen ✓ Only pipette mix immediately before use ✓ Short Fragment Buffer (SFB) ✓ ✓ Vortexing ✓ -
Select a unique barcode for each sample to be run together on the same flow cell.
Please note: Only use one barcode per sample.
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Using a stepper pipette, or a multichannel pipette, make up the Native Barcode 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(s) and spin down briefly.
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Incubate for 20 minutes at room temperature.
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Add EDTA to each well and mix thoroughly by pipetting and spin down briefly.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
Up to x24 samples x25 — x96 samples Volume of clear capped EDTA per sample 2 µl 1 µl Volume of blue capped EDTA per sample 4 µl 2 µl -
Pool the barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube:
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
x24 samples x48 samples x96 samples Total volume for preps using clear cap EDTA ~528 µl ~528 µl ~1,056 µl Total volume for preps using blue cap EDTA ~576 µl ~576 µl ~1,152 µl -
Resuspend the AMPure XP Beads (AXP) by vortexing.
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Add AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting for a 0.4X clean.
Volume for 10 µl of sample For 265 µl of samples For 528 µl of samples For 1,056 µl of samples Volume of AXP 4 µl 106 µl 211 µl 422 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 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. 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 by gently flicking.
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Incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Note: If stuggling to obtain the necessary yield, increasing the incubation period up to 30 minutes may improve elution efficacy.
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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.