- Materials
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- 200 ng of extracted gDNA per sample
- Rapid Barcodes (RB01-24) or Rapid Barcode Plate (RB01-96)
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
- AMPure XP Beads (AXP)
- Elution Buffer (EB)
- Consumables
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- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Ice bucket with ice
- Timer
- Thermal cycler
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Magnetic rack
- Hula mixer (gentle rotator mixer)
- Qubit fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
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Sample throughput and Rapid Barcode use requirements with NO-MISS
This method has been developed to process 24 samples with a genome size of up to 7 Mb simultaneously.
For samples with a larger genome size ( >7 Mb), we recommend lowering the number of samples to be barcoded and sequenced simultaneously to 8 samples.
For optimal output, we currently do not recommend using fewer than 4 barcodes.
Note: This method provides a standardised process for sample throughput that we have validated in-house. These settings will be applicable to the majority of use-cases and we recommend all new users to follow the recommended method. Experienced users can adjust sample throughput (4 to 48 barcoded samples) based on sample quality, genome size, and coverage requirements.
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Check your flow cell.
We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.
See the flow cell check instructions in the MinKNOW protocol for more information.
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Program the thermal cycler: 30°C for 2 minutes, then 80°C for 2 minutes.
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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 Rapid Barcodes (RB01-24) or Rapid Barcode Plate (RB01-96) Not frozen ✓ ✓ Rapid Adapter (RA) Not frozen ✓ ✓ AMPure XP Beads (AXP) ✓ ✓ Mix by pipetting or vortexing immediately before use Elution Buffer (EB) ✓ ✓ ✓ Adapter Buffer (ADB) ✓ ✓ Mix by vortexing -
Prepare the DNA in nuclease-free water.
- Transfer 200 ng of genomic DNA per sample into 0.2 ml thin-walled PCR tubes or an Eppendorf twin.tec® PCR plate 96 LoBind.
- Adjust the volume of each sample to 10 μl with nuclease-free water.
- Pipette mix the tubes thoroughly and spin down briefly in a microfuge.
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Select a unique barcode for every sample to be run together on the same flow cell.
Note: Use one barcode per sample.
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In the 0.2 ml thin-walled PCR tubes or an Eppendorf twin.tec® PCR plate 96 LoBind, mix the following:
Reagent Volume per sample Template DNA (200 ng from previous step) 10 μl Rapid Barcodes (RB01-24 or RB01-96, one for each sample) 1.5 μl Total 11.5 μl -
Ensure the components are thoroughly mixed by pipetting and spin down briefly.
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Incubate the tubes or plate at 30°C for 2 minutes and then at 80°C for 2 minutes. Briefly put the tubes or plate on ice to cool.
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Spin down the tubes or plate to collect the liquid at the bottom.
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Pool all barcoded samples in a clean 2 ml Eppendorf DNA LoBind tube, noting the total volume.
. Volume per sample For 24 samples Total volume 11.5 µl 276 µl -
Resuspend the AMPure XP Beads (AXP) by vortexing.
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Add an equal volume of resuspended AMPure XP Beads (AXP) to the entire pooled barcoded sample, and mix by flicking the tube.
. Volume per sample For 24 samples Volume of AMPure XP Beads (AXP) added 11.5 µl 276 µl -
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Prepare at least 2 ml of fresh 80% 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 1 ml of freshly prepared 80% 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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Briefly 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 the pellet by pipetting in 15 µl Elution Buffer (EB). Incubate for 10 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 the full volume of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
- Dispose of the pelleted beads
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Optional actionQuantify 1 µl of eluted sample using a Qubit fluorometer and Qubit dsDNA BR assay.
Expect ~150 ng/µl for 24 samples, assuming 70% of DNA was retained during the wash.
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Transfer 11 µl of the sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
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In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:
Reagent Volume Rapid Adapter (RA) 1.5 μl Adapter Buffer (ADB) 3.5 μl Total 5 μl -
Add 1 µl of the diluted Rapid Adapter (RA) 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.
Tip: While this incubation step is taking place you can proceed to the Flow Cell priming and loading section of the protocol.