- Materials
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- Native Barcodes (NB01-NB96)
- EDTA (EDTA)
- AMPure XP Beads (AXP)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Magnetic rack
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Microfuge
- Thermal cycler
- Ice bucket with ice
- 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 EDTA at room temperature and mix by vortexing. Then spin down and place on ice.
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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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Select a unique barcode for each sample to be run in a group:
- For two samples on one flow cell, select two unique barcodes, one for each sample.
- For three samples on two flow cells, select three unique barcodes, one for each sample.
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In clean 1.5 ml Eppendorf DNA LoBind tubes, add the reagents in the following order per sample:
Reagent Volume End-prepped DNA 7.5 µl Native barcode 2.5 µl Blunt/TA Ligase Master Mix 10 µl Total 20 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
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Incubate for 20 minutes at room temperature.
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Add 4 µl of EDTA to each tube and mix thoroughly by pipetting and spin down briefly.
Note: EDTA is added at this step to stop the reaction.
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Pool each group of two or three uniquely barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube.
For example:
- Up to 48 pools of two differentially barcoded samples
- Up to 32 pools of three differentially barcoded samples -
Ensure the beads are at room temperature and resuspend the AMPure XP beads (AXP) by vortexing.
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Add AMPure XP Beads (AXP) to the pooled reaction in a 0.4X ratio and mix by pipetting. The volume for AMPure XP Beads (AXP) will vary depending on the number of barcoded samples in the pool:
- For two barcoded samples add 19 µl of AMPure XP Beads (AXP)
- For three barcoded samples add 29 µl of AMPure XP Beads (AXP)
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Prepare 1 ml of fresh 80% ethanol in nuclease-free water per barcoded sample pool.
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Spin down the sample for 5 seconds and pellet on a magnet for 5 minutes. Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.
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Keep the tube on the magentic rack and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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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 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.
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Spin down the tube for 5 seconds and 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.