- Materials
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- ~ 3.5 µg fragmented DNA in 45 µl
- PCR Adapter (PCA)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Hula mixer (gentle rotator mixer)
- Qubit fluorometer (or equivalent for QC check)
- Microfuge
- Vortex mixer
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Add the reagents in the order given below, mixing by pipetting 10-20 times between each sequential addition:
Reagent Volume End-prepped DNA 30 µl PCR Adapters (PCA) 20 µl NEB Blunt/TA Ligase Master Mix 50 µl Total 100 µl -
Mix gently by flicking the tube, and spin down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 100 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
End-prep cleanup demo
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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. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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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 pellet in 49 µl nuclease-free water. 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 49 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of adapted DNA using a Qubit fluorometer.