- Materials
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- Primer Mix (PRM)
- Consumables
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- 0.2 ml thin-walled PCR tubes
- 2x Long AMP Taq
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Equipment
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- Thermal cycler
- Ice bucket with ice
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Hula mixer (gentle rotator mixer)
- Vortex mixer
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In a 0.2 ml thin-walled PCR tube mix the following:
Reagent Volume 2x Long AMP Taq 50 µl PRM Adapters (10 μM) 2 µl Template DNA 48 µl Total 100 µl -
Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 3 mins 1 Denaturation 98 °C 20 secs 11 (b) Annealing 62 °C (a) 15 secs (a) 11 (b) Extension 65 °C (c) 3 mins 11 (b) Final extension 65 °C 3 mins 1 Hold 4 °C ∞ a. This is specific to the Oxford Nanopore primer and should be maintained
b. Adjust accordingly if input quantities are altered
c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)
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Resuspend the AMPure XP beads by vortexing.
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Add 180 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
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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 50 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 50 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 2 µl of amplified DNA using a Qubit fluorometer.