- Materials
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- 200 ng 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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Minimum Rapid Barcode use requirements
For optimal output, we currently do not recommend using fewer than 4 barcodes. If you wish to multiplex less than 4 samples, please ensure you split your sample(s) across barcodes so a minimum of 4 barcodes are run:
- For 1 sample, run your sample across 4 barcodes (e.g. RB01-RB04 using 200ng of sample A per barcode)
- For 2 samples, run each sample across two barcodes. (e.g. RB01-RB02 for sample A and RB03-RB04 for sample B)
- For 3 samples, run two samples individually and one across 2 barcodes. (e.g. RB01 and RB02 for sample A and B respectively, and RB03-RB04 for sample C)
Please note that the required sample input for each barcode is 200 ng gDNA.
Alternatively, you might want to explore our Rapid Sequencing Kit V14 (SQK-RAD114) for sequencing individual or small sets of samples.
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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 for 10-15 times to avoid unwanted shearing
- Spin down briefly in a microfuge
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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 4 samples For 12 samples For 24 samples For 48 samples For 96 samples Total volume 11.5 µl 46 µl 138 µl 276 µl 552 µl 1,104 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 4 samples For 12 samples For 24 samples For 48 samples For 96 samples Volume of AMPure XP Beads (AXP) added 11.5 µl 46 µl 138 µl 276 µl 552 µl 1,000 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 in 15 µl Elution Buffer (EB) per 24 barcodes used.
. For 24 barcodes For 48 barcodes For 72 barcodes For 96 barcodes Volume of Elution Buffer (EB) 15 µl 30 µl 45 µl 60 µl -
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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Quantify 1 µl of eluted sample using a Qubit fluorometer.
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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.