- Materials
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- PCR Barcode Primers (BC01-12, at 10 µM)
- Consumables
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- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Equipment
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- Thermal cycler
- Magnetic rack
- Hula mixer (gentle rotator mixer)
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Set up a barcoding PCR reaction as follows for each library:
The following is written for LongAmp Taq, but can be adapted for use with other polymerases.
Between each addition, pipette mix 10-20 times.
Reagent Volume PCR Barcode (one of BC01-BC12, at 10 µM) 2 µl 10 ng/µl adapter ligated template 2 µl LongAmp Taq 2x master mix 50 µl Nuclease-free water 46 µl Total volume 100 µl -
If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.
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Mix gently by flicking the tube, and spin down.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 3 mins 1 Denaturation 95 °C 15 secs 12-15 (b) Annealing 62 °C (a) 15 secs (a) 12-15 (b) Extension 65 °C (c) dependent on length of target fragment (d) 12-15 (b) Final extension 65 °C dependent on length of target fragment (d) 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)
d. Adjust accordingly for different lengths of amplicons and the type of polymerase that is being used. Oxford Nanopore R&D teams standardly use 8 min for DNA fragmented to 8 kb.
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Transfer each sample to a separate 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 each 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 sufficient 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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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 each pellet in 10 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 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Dispose of the pelleted beads
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Quantify the barcoded library using standard techniques, and pool all barcoded libraries in the desired ratios in a 1.5 ml DNA LoBind Eppendorf tube.
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Prepare 1 µg of pooled barcoded libraries in 47 µl nuclease-free water.