- Materials
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- 10 ng high molecular weight genomic DNA
- 16S Barcodes in 96-well plate, at 1 μM each
- Rapid Adapter (RAP)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Ice bucket with ice
- Microfuge
- Multichannel pipette and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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Take one 96-well plate containing 16S barcodes. Break one set of barcodes (1-24, or as desired) away from the plate and return the rest to storage.
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Thaw the desired barcodes, make sure the liquid is at the bottom of the tubes, and place on ice.
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Thaw the LongAmp Hot Start Taq 2X Master Mix, spin down briefly, mix well by pipetting and place on ice.
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Prepare the DNA in nuclease-free water.
- Transfer 10 ng genomic DNA into a DNA LoBind tube
- Adjust the volume to 10 μl with nuclease-free water
- Mix thoroughly by flicking the tube, to avoid unwanted shearing
- Spin down briefly in a microfuge
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For each sample to be tested, prepare the following mixture in separate 0.2 ml thin-walled PCR tubes.
Reagent Volume Nuclease-free water 5 µl Input DNA (10 ng) 10 µl LongAmp Hot Start Taq 2X Master Mix 25 µl Total 40 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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Ensure the components are thoroughly mixed by pipetting, and spin down.
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Using clean pipette tips, carefully pierce the foil surface of the required barcodes. Use a new tip for each barcode to avoid cross-contamination. Make a note of which barcode numbers will be run for each sample.
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Using a multichannel pipette, mix the 16S barcodes by pipetting up and down 10 times. Transfer 10 μl of each 16S Barcode into respective sample-containing tubes.
The layout of the barcodes in the plate are as follows:
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Mix thoroughly by pipetting up and down ten times.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 1 min 1 Denaturation 95 °C 20 secs 25 Annealing 55 °C 30 secs 25 Extension 65 °C 2 mins 25 Final extension 65 °C 5 mins 1 Hold 4 °C ∞ -
Transfer each sample to a separate 1.5 ml DNA LoBind Eppendorf tube. Carry out steps 11-21 for each sample, before pooling the samples at step 22.
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Resuspend the AMPure XP beads by vortexing.
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Add 30 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 10 µl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. 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 1 µl of eluted sample using a Qubit fluorometer.
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Pool all barcoded libraries in the desired ratios to a total of 50-100 fmoles in 10 μl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. For 16S amplicons of ~1500 bp, 50-100 fmoles equates to ~50-100 ng.
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Add 1 μl of RAP to the barcoded DNA.
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Mix gently by flicking the tube, and spin down.
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Incubate the reaction for 5 minutes at room temperature.
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Optional actionIf quantities allow, the libraries may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.
Additional Elution Buffer (EB) for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately.