- Materials
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- Rapid Barcode Primers (RLB01-12A, at 10 µM)
- Fragmentation Mix (FRM)
- Rapid Adapter (RAP)
- Consumables
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- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl (e.g. Fisher Scientific, Cat# NC1877767)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Microfuge
- Thermal cycler
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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DNA tagmentation
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Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.
Reagent 1. Thaw 2. Mix well by pipetting 3. Briefly spin down Rapid Barcode Primers (RBL 01-12A) At room temperature ✓ ✓ Fragmentation Mix (FRM) Not frozen ✓ ✓ Rapid Adapter Not frozen ✓ ✓ -
Prepare the LongAmp Taq 2X Master Mix according to the manufacturer's recommendations and prepare at least 20 µl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl.
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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 and clean-up
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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 pipetting 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 30 Annealing 56°C 15 secs 30 Extension 65°C 4 mins 30 Final extension 65°C 4 mins 1 Hold 4°C ∞ -
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 attachment
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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.