- Materials
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- 1000 ng gDNA per sample
- Consumables
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- NEBNext FFPE DNA Repair Mix (NEB, cat # M6630)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Ice bucket with ice
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Qubit fluorometer (or equivalent)
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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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In clean 0.2 ml thin-walled PCR tubes, aliquot 1000 ng per sample.
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Make up each sample to 12 µl using nuclease-free water. Mix gently by pipetting and spin down.
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Combine the following components per sample:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume NEBNext FFPE DNA Repair Buffer 0.875 µl Ultra II End-prep reaction buffer 0.875 µl Ultra II End-prep enzyme mix 0.75 µl NEBNext FFPE DNA Repair Mix 0.50 µl Total 3 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.
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Transfer each sample to clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the Agencourt AMPure XP beads by vortexing.
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Add 15 µl of resuspended Agencourt AMPure XP beads to each 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 samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes 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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Briefly spin down and place the tubes 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 tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 10 µl of eluate for each sample into clean 1.5 ml Eppendorf DNA LoBind tubes, individually.
Note: If users are having difficulty retaining 10 µl without disturbing the beads, 8.5 µl can be retained instead, allowing 1 µl for quantification and 7.5 µl to be taken forward into the Native Barcode Ligation step.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.