- 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)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 0.2 ml 96-well PCR plate
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Thermal cycler
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Capping and decapping the 96 well format
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Layout of barcodes in the 96 tube plate
The wells of the 96 tube plate correspond to the barcodes in the following way. All barcodes are supplied at 10 µM concentration and to be used at a final concentration of 0.2 µM.
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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 BC1-BC96, at 10 µM) 1 µl Adapter-ligated DNA 2 µl LongAmp Taq 2x master mix 25 µl Nuclease-free water 22 µl Total volume 50 µl -
The amount of input DNA may need to be adjusted depending on application. For example, for sequencing human or larger genomes, we recommend putting ~50 ng DNA into a PCR reaction. For amplicons or smaller genomes, the 20 ng stated above is sufficient. 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 by pipetting.
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Seal the plate with adhesive film or PCR strip caps and briefly spin down in a plate spinner.
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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 15-18 (b) Annealing 62 °C (a) 15 secs (a) 15-18 (b) Extension 65 °C (c) dependent on length of target fragment (d) 15-18 (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 (LongAmp Taq amplifies at a rate of 50 seconds per kb)
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Resuspend the AMPure XP beads by vortexing.
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Add 35 µl of of resuspended AMPure XP beads to each sample and mix by pipetting the entire combined volume up and down 10 times.
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Incubate for 5 minutes at room temperature.
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Prepare sufficient fresh 70% ethanol in nuclease-free water.
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Pellet the beads on a magnet for at least 2 min, or until the supernatant is clear. Keep the plate on the magnet and pipette off the supernatant.
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Wash each pellet of beads by adding 200 µl of freshly-prepared 70% ethanol. Resuspend each pellet thoroughly by pipetting the entire volume of buffer up and down ten times. Return the plate to the magnetic rack and allow the beads to pellet until the supernatant is clear. Remove the supernatant using a pipette and discard.
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Repeat the previous step.
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Seal the plate. Spin down and place the plate back on the magnet. Pipette off any residual supernatant.
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Remove the plate from the magnetic rack and resuspend each pellet in 21 µl nuclease-free water, pipetting the entire volume up and down 10 times. 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 21 µl of each eluate into a well of a clean 96-well plate.
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Quantify 1 µl of each barcoded DNA sample using a Qubit fluorometer.
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Optional actionIf your DNA samples are of mixed sizes, we recommend either running a subset of samples on an Agilent Bioanalyzer, or estimating the DNA size from a gel.
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Using the DNA mass (calculated using the Qubit fluorometer) and size distribution (calculated using a gel or Agilent Bioanalyzer), pool equimolar quantities of barcoded amplicons in batches of 96, ensuring that every sample within a given pool has a unique barcode.
Using the DNA mass (calculated using the Qubit fluorometer) and size distribution (calculated using a gel or Agilent Bioanalyzer), pool equimolar quantities of barcoded amplicons in batches of 96 or fewer, depending on how many of the 96 barcodes were used. Ensure that every sample within a given pool has a unique barcode.