- 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)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml PCR tubes
- 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
- 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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Select a unique barcode for each sample to be run together on the same flow cell.
Note: Only use one barcode per sample.
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In clean 0.2 ml PCR-tubes, add the reagents in the following order per well:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume End-prepped rAAV 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.
Volume per sample For 6 samples Total volume for preps using clear cap EDTA 22 µl 132 µl Total volume for preps using blue cap EDTA 24 µl 144 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.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
/ Volume per sample For 6 samples Volume of AXP for preps using clear cap EDTA 9 µl 53 µl Volume of AXP for preps using blue cap EDTA 10 µl 58 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 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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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 containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
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Quantify 1 µl of eluted sample using a Qubit fluorometer.