- Materials
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- Extracted rAAV DNA in 50 µl per sample
- AMPure XP Beads (AXP)
- Consumables
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- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Qubit dsDNA HS Assay Kit (Invitrogen, 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 thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Multichannel pipette and tips
- Thermal cycler
- Microfuge
- Ice bucket with ice
- Magnetic rack
- Vortex mixer
- Hula mixer (rotator mixer)
- Qubit fluorometer (or equivalent)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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Check your flow cell.
We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.
See the flow cell check instructions in the MinKNOW protocol for more information.
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Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing. Keep the beads at room temperature until use.
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Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice:
For optimal perfomance, NEB recommend the following:
Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
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If the optional annealing step was skipped, make up each AAV sample to 50 µl with nuclease-free water.
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For each rAAV sample, combine the following reagents in a 0.2 ml PCR tube.
Between each addition, pipette mix 10-20 times.
Reagent Volume rAAV DNA 50 µl NEBNext Ultra II End-prep Reaction Buffer 7 µl NEBNext Ultra II End-prep Enzyme Mix 3 µl Total 60 µl -
Ensure the components are thoroughly mixed by pipetting and spin down briefly.
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Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
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Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the AMPure XP beads (AXP) by vortexing.
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Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
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Incubate the samples on a Hula Mixer (rotator mixer) for 10 minutes at room temperature.
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Prepare sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
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Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 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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Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
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Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water.
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Incubate the samples on a Hula Mixer (rotator mixer) for 10 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.