- Materials
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- ~400 ng plasmid DNA per sample
- Fragmentation Mix RB01-12
- Rapid Adapter (RAP)
- Consumables
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- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl (e.g. Fisher Scientific, Cat# NC1877767)
- Freshly prepared 70% ethanol in nuclease-free water
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Equipment
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- Thermal cycler or heat blocks
- Microfuge
- P10 pipette and tips
- Optional equipment
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- Magnetic rack
- Hula mixer (gentle rotator mixer)
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Thaw kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:
Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting Fragmentation Mix RB01-12 Not frozen ✓ ✓ Rapid Adapter (RAP) Not frozen ✓ ✓ Sequencing Buffer (SQB) ✓ ✓ ✓* Loading Beads (LB) ✓ ✓ Mix by pipetting or vortexing immediately before use Flush Buffer (FLB) - 1 tube ✓ ✓ ✓* Flush Tether (FLT) ✓ ✓ ✓ *Vortexing, followed by a brief spin in a microfuge, is recommended for Sequencing Buffer (SQB) and Flush Buffer (FLB).
Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol. It is provided in the kit for potential future product compatibility.
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Prepare the DNA in nuclease-free water.
- ~400 ng of plasmid DNA is required in 7.5 µl of volume for each sample for barcoding; see table below for dilutions:
Starting Conc. Volume of DNA Volume of nuclease-free water Total volume 1000 ng/µl 1 µl 19 µl 20 µl 800 ng/µl 2 µl 28 µl 30 µl 600 ng/µl 4 µl 41 µl 45 µl 200 ng/µl 8 µl 22 µl 30 µl 100 ng/µl 7 µl 6 µl 13 µl 90 ng/µl 16 µl 11 µl 27 µl 70 ng/µl 10 µl 3 µl 13 µl <53 ng/µl 7.5 µl 0 µl 7.5 µl - Pipette mix the dilutions, and spin down briefly in a microfuge.
- Take forward 7.5 μl of the diluted plasmid sample.
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In a 0.2 ml thin-walled PCR tube, mix the following:
Reagent Volume 400 ng template DNA 7.5 μl Fragmentation Mix RB01-12 (one for each sample) 2.5 μl Total 10 μl -
Pipette mix and spin down
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Incubate the tube at 30°C for 1 minute and then at 80°C for 1 minute. Briefly put the tube on ice to cool it down.
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Pool all barcoded samples into a single 1.5 ml Eppendorf DNA LoBind tube, noting the total volume.
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Resuspend the AMPure XP beads by vortexing.
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To the entire pooled barcoded sample from Step 6, add an equal volume of resuspended AMPure XP beads, and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Top tip: This is a good time to check your flow cell while you wait. See the instructions for checking your flow cell on MinION.
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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.
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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 70% ethanol. Briefly allow to dry.
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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, for at least 1 minute.
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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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Add 1 µl of RAP to 10 µl of barcoded DNA.
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Pipette mix and spin down
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Incubate the reaction for 5 minutes at room temperature.