- Materials
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- 100 ng of each sheared DNA sample to be barcoded in 45 µl
- Consumables
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- NEBNext® Ultra II End Prep Enzyme Mix from NEBNext® Ultra II End Repair Module (NEB, E7546)
- NEBNext® Ultra II End Prep Reaction Buffer from NEBNext® Ultra II End Repair Module (NEB, E7546)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- 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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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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Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice:
For optimal performance, NEB recommend the following:
Thaw all reagents on ice.
Ensure the reagents 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 NEBNext Ultra II End Prep Reaction Buffer may contain a white precipitate. If this occurs, allow the mixture(s) to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.
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Prepare the DNA in nuclease-free water.
- Transfer 100 ng 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:
Reagent Volume per sample 100 ng 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.