- Materials
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- Pooled ASFV amplicons
- Consumables
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- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- NEBNext FFPE DNA Repair Mix (NEB, M6630)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
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Prepare the NEBNext FFPE DNA Repair Mix and 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.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer and FFPE DNA Repair 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.
Note: It is important the buffers are mixed well by vortexing.The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.
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Prepare the DNA in nuclease-free water.
- Transfer 700 ng of amplicon DNA into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to 48 μl with nuclease-free water
- Mix thoroughly by flicking the tube
- Spin down briefly in a microfuge
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In a 0.2 ml thin-walled PCR tube, mix the following:
Between each addition, pipette mix 10-20 times
Reagent Volume DNA 48 µl NEBNext FFPE DNA Repair Buffer 3.5 µl Ultra II End-prep reaction buffer 3.5 µl Ultra II End-prep enzyme mix 3 µl NEBNext FFPE DNA Repair Mix 2 µl Total 60 µl -
Mix well by pipetting and spin down.
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Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
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Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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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 flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% 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 tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the tube from the magnetic rack and resuspend the pellet in 25 µl nuclease-free water. Spin down and incubate for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
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Remove and retain 25 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Optional actionQuantify 1 µl of eluted sample using a Qubit fluorometer.