- Materials
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- 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded
- DNA Control Sample (DCS)
- Consumables
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- 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
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Equipment
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- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
- Thermal cycler
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Ice bucket with ice
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Thaw the DNA Control Sample (DCS) at room temperature, mix by vortexing, and place on 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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Dilute your DNA Control Sample (DCS) by adding 105 µl Elution Buffer (EB) directly to one DCS tube. Mix gently by pipetting and spin down.
One tube of diluted DNA Control Sample (DCS) is enough for 140 samples. Excess can be stored at -20°C in the freezer.
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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 to 11.5 µl using nuclease-free water. Mix gently by pipetting and spin down.
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Combine the following components per well:
Between each addition, pipette mix 10-20 times.
Reagents Volume 200 fmol amplicon DNA 11.5 µl Diluted DNA Control Sample (DCS) 1 µl Ultra II End-prep Reaction Buffer 1.75 µl Ultra II End-prep Enzyme Mix 0.75 µl Total 15 µl -
Ensure the components are thoroughly mixed by pipetting and spin down briefly.
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Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.