- 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 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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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.