- Materials
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- 1–5 ng high molecular weight genomic DNA
- Fragmentation Mix (FRM)
- Rapid Adapter (RAP)
- Rapid Barcode Primers (RLB01-12A, at 10 µM)
- Consumables
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- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
- 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
- P10 pipette and tips
- P2 pipette and tips
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Thaw and prepare the reagents as follows:
Contents On ice At room temperature Barcodes (RLB 01-12A) ✓ Fragmentation Mix (FRM) ✓ Rapid Adapter (RAP) ✓ Once thawed, mix the RLB barcodes by pipetting up and down, and spin down briefly. The barcodes should now be stored on ice.
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DNA tagmentation
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Prepare the DNA in nuclease-free water.
- Transfer 1-5 ng genomic DNA into a DNA LoBind tube
- Adjust the volume to 3 μl with nuclease-free water
- Mix thoroughly by flicking avoiding unwanted shearing
- Spin down briefly in a microfuge
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In a 0.2 ml thin-walled PCR tube, mix the following:
Reagent Volume 1-5 ng template DNA 3 μl Fragmentation Mix (FRM) 1 μl Total 4 μl -
Mix gently by flicking the tube, and spin down.
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In a thermal cycler, 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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PCR
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Set up a PCR reaction as follows in a 0.2 ml thin-walled PCR tube:
Reagent Volume Nuclease-free water 20 µl Tagmented DNA 4 µl RLB (01-12A, at 10 µM) 1 µl LongAmp Taq 2X master mix 25 µl Total 50 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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Mix gently by flicking the tube, and spin down.
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Amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 95 °C 3 mins 1 Denaturation 95 °C 15 secs 14 Annealing 56 °C 15 secs 14 Extension 65 °C 6 mins 14 Final extension 65 °C 6 mins 1 Hold 4 °C ∞ Adjust the extension time accordingly for different lengths of amplicons and the type of polymerase that is being used.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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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.
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Adapter ligation
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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.