- Materials
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- PCR Barcodes (BC01-96, 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
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- OR 0.2 ml thin-walled PCR tubes
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
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- Thermal cycler
- Magnetic rack
- Microfuge
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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Please note, this protocol is written for a template input of 100–200 fmol with PCR Barcodes (BC01-96) used at a final concentration of 0.2 µM. However, the input mass and the number of PCR cycles may be adjusted as appropriate depending on the requirements of the experiment.
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Thaw the PCR Barcodes (BC01-96) required for your number of samples at room temperature. Individually mix the barcodes by pipetting, spin down, and place on ice.
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Prepare the samples in nuclease-free water:
- Transfer 100-200 fmol of each sample to a clear 0.2 ml PCR tube or plate
- For 1–12 samples: Adjust the volume to 48 μl with nuclease-free water
- For 13–96 samples: Adjust the volume to 24 μl with nuclease-free water
- Mix thoroughly by flicking the tube or plate to avoid unwanted shearing
- Spin down briefly in a microfuge
- Transfer 100-200 fmol of each sample to a clear 0.2 ml PCR tube or plate
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Select a unique barcode for each sample to be processed in the PCR barcoded pool.
Note: Only use one barcode per sample.
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Set up a barcoding PCR reaction as follows for each library in fresh 0.2 ml PCR tubes or a 0.2 ml 96-well PCR plate.
Between each addition, pipette mix 10-20 times.
Reagent Volume per sample for using 1–12 barcodes Volume per sample for using 13 barcodes or more PCR Barcode (one of BC1-BC96, at 10 µM) 2 µl 1 µl Adapter-ligated DNA 48 µl 24 µl LongAmp Taq 2x master mix 50 µl 25 µl Total volume 100 µl 50 µl -
Mix by pipetting and briefly 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. During the development of this kit, 8 minutes was used as standard for DNA fragmented to 8 kb.
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Resuspend the AMPure XP beads by vortexing.
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Add 0.4X volume of resuspended AMPure XP Beads to each reaction and mix by flicking the tube.
Reagent Volume for 100 µl samples Volume for 50 µl samples AMPure XP Beads 40 µl 20 µl -
Incubate at room temperature for 5 minutes.
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Prepare sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
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Place samples on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Keep the samples on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellets. 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 samples back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellets to the point of cracking.
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Remove the samples 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 magnetic rack until the eluate is clear and colourless.
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Remove and retain 10 µl of each eluate into clean 0.2 ml PCR tubes or a clean PCR plate.
- Dispose of the pelleted beads
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Quantify the PCR barcoded samples using a Qubit fluorometer and pool all barcoded samples in the desired ratios into a 1.5 ml DNA LoBind Eppendorf tube for each PCR barcoded sample pool.
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Prepare each pooled PCR barcoded sample pool to 200 fmol (130 ng for 1 kb amplicons) in separate tubes and make the volume up to 12.5 µl nuclease-free water.
If the volume of a pool exceeds the 12.5 µl required for the end-prep reaction, consider a 2.5X AMPure XP Bead purification of the pool to concentrate your sample.