- Materials
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- Native Barcodes (NB01-24)
- AMPure XP Beads (AXP)
- EDTA (EDTA)
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- OR 0.2 ml thin-walled PCR tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Equipment
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- Magnetic rack
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Microfuge
- Thermal cycler
- Ice bucket with ice
- Multichannel pipette and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- 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 (NB01-24) required for your number of samples at room temperature. Individually mix the barcodes by pipetting, spin down, and place them on ice.
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Normalise and prepare your end-prepped cfDNA samples in nuclease-free water:
Inputs should be normalised so the same mass of sample is used in each reaction.
For each sample, take forward an equivalent mass of sample into a separate clean 0.2 ml thin-walled PCR tube.
Note: To take forward the maximum amount of DNA possible, we recommend taking the full volume of your lowest concentration sample and normalising the other samples to to this.Adjust the volume of each sample to 7.5 μl with nuclease-free water.
Mix thoroughly by pipetting up and down, or by flicking the tube(s).
Spin down briefly in a microfuge.
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Select a unique barcode for each sample to be run together on the same flow cell. 12 samples should be barcoded and combined in one experiment.
Please note: Only use one barcode per sample.
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In the 0.2 ml PCR-tubes containing your normalised sample inputs, add the reagents in the following order for each sample:
Reagent Volume End-prepped DNA 7.5 µl Native Barcode (NB01-24) 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 the following volume of 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.
EDTA cap colour Volume per well For clear cap EDTA 2 µl For blue cap EDTA 4 µl -
Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
. For 12 samples Total volume for preps using clear cap EDTA 264 µl Total volume for preps using blue cap EDTA 288 µl -
Resuspend the AMPure XP Beads (AXP) by vortexing.
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Add 1.2X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
. For 12 samples Volume of AXP for preps using clear cap EDTA 317 µl Volume of AXP for preps using blue cap EDTA 346 µl -
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
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Spin down the sample 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 magnetic rack and wash the beads with 700 µl of freshly prepared 80% 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 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 32 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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Pellet the beads on a magnetic rack until the eluate is clear and colourless.
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Remove and retain 32 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.
Note: You should expect to recover between 40–60 % of the input DNA mass. For example, if starting with 6 ng per sample and using 12x barcodes (72 ng total), a yield of approximately 30–40 ng, is expected.