- Materials
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- 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- Equipment
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- Multichannel pipette and tips
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Microfuge
- Ice bucket with ice
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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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In a clean 96-well plate, aliquot 200 fmol (130 ng for 1 kb amplicons) of DNA per sample.
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Make up each sample per well to 12.5 µl using nuclease-free water.
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Add the following components to each well:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume Ultra II End-prep reaction buffer 1.75 µl Ultra II End-prep enzyme mix 0.75 µl Total 2.5 µl -
Mix well by pipetting and spin down in a centrifuge.
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Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.