- Materials
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- DNA library eluate
- Universal amplification mastermix
- Consumables
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- 0.2 ml thin-walled PCR tubes
- 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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- Microfuge
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack suitable for 0.2 ml PCR tubes
- Qubit fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P100 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Vortex mixer
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In a clean 0.2 ml PCR tube, set up the following reaction:
Reagent Volume DNA library eluate 18 µl Universal amplification mastermix 32 µl Total 50 µl -
Mix gently by flicking the tube and spin down.
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Incubate using the following program:
Step Temperature Ramp rate Time Cycles Initial denaturation 98°C max 3 min 1 Denaturation
Extension98°C
72°Cmax
max20 sec
2 min25 Final extension 72°C max 5 min 1 Hold 4°C max - - Note: if a yield of >200 fmols is not achieved, please optimise the amplification with additional cycles as required in future experiments.
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Resuspend the AMPure XP beads by vortexing.
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Add 45 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 10 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 the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear, 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 30 µl of nuclease-free water. Incubate for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear.
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Remove and retain 30 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.