- Materials
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- 50 ng of high molecular weight plasmid DNA 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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- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- 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)
- 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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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, as follows. Approximately 50 ng of plasmid DNA is required in 9 µl of volume for each sample for barcoding.
- Dilute your plasmid DNA samples with nuclease-free water to approximately 50 ng. See the table below for dilutions:
Starting Conc. Volume of DNA Volume of nuclease-free water Total volume 100 ng/µl 2 µl 34 µl 36 µl 90 ng/µl 2 µl 31 µl 33 µl 80 ng/µl 2 µl 27 µl 29 µl 70 ng/µl 3 µl 35 µl 38 µl 60 ng/µl 2 µl 20 µl 22 µl 50 ng/µl 2 µl 16 µl 18 µl 40 ng/µl 5 µl 31 µl 36 µl 30 ng/µl 5 µl 22 µl 27 µl 20 ng/µl 5 µl 13 µl 18 µl 10 ng/µl 10 µl 8 µl 18 µl <5.56 ng/µl 9 µl 0 µl 9 µl - Pipette mix the dilutions, and spin down briefly.
- Add 9 µl of volume for each sample into a 0.2 ml PCR tube or plate.
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Select a unique barcode for every sample to be run together on the same flow cell. Up to 96 samples can be barcoded and combined in one experiment.
Please note: Only use one barcode per sample.
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In 0.2 ml thin-walled PCR tubes or plate, mix the following reagents. The Rapid Barcodes can be transferred using a multichannel pipette:
Reagent Volume 50 ng template DNA 9 μl Rapid Barcodes (RB01-96, one for each sample) 1 μl Total 10 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 the barcoded samples into a clean 1.5 ml Eppendorf DNA LoBind tube, noting the total volume.
. Volume per sample For 12 samples For 24 samples For 48 samples For 96 samples Total volume 10 μl 120 μl 240 μl 480 μl 960 μl -
Resuspend the AMPure XP beads (AXP) by vortexing.
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To the entire pooled barcoded sample, add an equal volume of resuspended AMPure XP Beads (AXP) and mix by flicking the tube.
. Volume per sample For 12 samples For 24 samples For 48 samples For 96 samples Volume of AXP 10 μl 120 μl 240 μl 480 μl 960 μl -
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare at least 3 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 1.5 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). 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 15 µl 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.
Note: We recommend transfering a maximum of 800 ng of the DNA library.
If necessary, take forward only the necessary volume for 800 ng of DNA library and make up the rest of the volume to 11 µl using Elution Buffer (EB). -
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.