- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
- Equipment
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- Multichannel pipette capable of dispensing 0.5 – 10 µL, and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Microfuge
- Ice bucket with ice
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Vortex mixer
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Determine the volume of the cleaned-up PCR reaction that yields 200 fmol of DNA per sample and aliquot into a clean 96-well plate (End-prep plate).
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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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Make up each sample per well to 12.5 µl using nuclease-free water.
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Prepare the following end-prep master mix in 1.5 ml Eppendorf DNA LoBind tube and mix thoroughly by pipetting:
Reagent Volume per reaction For X24 samples For X48 samples For X96 samples Ultra II End-prep reaction buffer 1.75 µl 52.5 µl 105 µl 210 µl Ultra II End-prep enzyme mix 0.75 µl 22.5 µl 45 µl 90 µl Total 2.5 µl 75 µl 150 µl 300 µl -
Using a stepper pipette or multi-channel pipette, add 2.5 µl of the end-prep master mix to each well containing 12.5 µl sample.
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Ensure the reactions are thoroughly mixed by pipetting. Seal the End-prep plate and spin down briefly.
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Using a thermal cycler, incubate the plate at 20°C for 5 minutes and 65°C for 5 minutes.