- Materials
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- <100–200 fmol of each DNA sample to be barcoded in 45 µl
- Consumables
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- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- 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 (Invitrogen, Q32851)
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Ice bucket with ice
- Microfuge
- Magnetic rack
- Vortex mixer
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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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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Prepare the DNA in nuclease-free water.
- Transfer <100-200 fmol DNA of each sample into a fresh 0.2 ml PCR tube or plate
- Adjust the volume to 45 μl with nuclease-free water
- Mix thoroughly by flicking the tube to avoid unwanted shearing
- Spin down briefly in a microfuge
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Set up the end-repair reaction as follows for each library:
Between each addition, pipette mix 10-20 times.
Reagent Volume per sample <100-200 fmol DNA 45 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Nuclease-free water 5 µl Total 60 µl -
Mix by pipetting and briefly spin down.
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Using a thermal cycler, incubate for 5 minutes at 20 °C and 5 minutes at 65 °C.
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Resuspend the AMPure XP beads by vortexing.
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Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
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Incubate at room temperature for 5 minutes.
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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 on a magnet until supernatant is clear and colourless. Keep the samples on the magnet, and pipette off the supernatant.
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Keep the samples 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.
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Repeat the previous step.
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Spin down and place the samples 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 samples from the magnet and resuspend each pellet in 16 µl nuclease-free water. Incubate for 2 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 eluate once it is clear and colourless. Transfer each eluted sample to a new tube or plate well.
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Quantify 1 µl of end-prepped DNA using a Qubit fluorometer - recovery aim <100–200 fmol.