- Materials
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- 1–5 ng high molecular weight genomic DNA
- Fragmentation Mix (FRM)
- Rapid Barcode Primers (RLB01-24, at 10 µM)
- EDTA (EDTA)
- AMPure XP Beads (AXP)
- Elution Buffer (EB)
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Ice bucket with ice
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Microfuge
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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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 or vortexing Rapid Barcode Primers (RLB01-24) Not frozen ✓ Pipette Fragmentation Mix (FRM) Not frozen ✓ Pipette Rapid Adapter (RA) Not frozen ✓ Pipette Adapter Buffer (ADB) ✓ ✓ Vortex or Pipette AMPure XP Beads (AXP) ✓ ✓ Mix by pipetting or vortexing immediately before use Elution Buffer (EB) ✓ ✓ Vortex or Pipette EDTA (EDTA) ✓ ✓ Vortex or Pipette Note: Once thawed, keep all kit components on ice.
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Prepare the DNA in nuclease-free water.
- Transfer 1–5 ng of each genomic DNA sample into a 1.5 ml Eppendorf 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 2 minutes and then at 80°C for 2 minutes. Briefly put the tube on ice to cool it down.
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For each sample, 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 (01-24, 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
Annealing
Extension95°C
56°C
65°C15 sec
15 sec
6 min
14Final extension 65°C 6 min 1 Hold 4°C ∞ -
Add 4 µl of EDTA to each barcoded sample, mix thorougly by pipetting and spin down briefly.
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Incubate for 5 minutes at room temperature.
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Quantify 1 µl of each barcoded sample using a Qubit fluorometer (or equivalent) for QC check.
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Pool all barcoded samples in equimolar ratios to a combined final concentration of 200–400 fmol (~400–800 ng) in a 1.5 ml Eppendorf DNA LoBind tube.
For a 200 fmol final pool:
Number of barcoded samples pooled 2 samples 6 samples 12 samples 24 samples Concentration of each barcoded sample added to the pool 100 fmol
(~200 ng)33.3 fmol
(~66.7 ng)16.7 fmol
(~33.3 ng)8.3 fmol
(~16.7 ng)Final pool concentration 200 fmol
(~400 ng)200 fmol
(~400 ng)200 fmol
(~400 ng)200 fmol
(~400 ng)For a 300 fmol final pool:
Number of barcoded samples pooled 2 samples 6 samples 12 samples 24 samples Concentration of each barcoded sample added to the pool 150 fmol
(~300 ng)50 fmol
(~100 ng)25 fmol
(~50 ng)12.5 fmol
(~25 ng)Final pool concentration 300 fmol
(~600 ng)300 fmol
(~600 ng)300 fmol
(~600 ng)300 fmol
(~600 ng)For a 400 fmol final pool:
Number of barcoded samples pooled 2 samples 6 samples 12 samples 24 samples Concentration of each barcoded sample added to the pool 200 fmol
(~400 ng)66.7 fmol
(~133.3 ng)33.3 fmol
(~66.7 ng)16.7 fmol
(~33.3 ng)Final pool concentration 400 fmol
(~800 ng)400 fmol
(~800 ng)400 fmol
(~800 ng)400 fmol
(~800 ng)Note: Please ensure you have quantified your samples prior to this step and take forward an equimolar concentration of each of the samples for optimal barcode balancing.
Samples may vary in concentration following the barcoded PCR, therefore the volume of each barcoded sample added to the pool will be different. -
Resuspend the AMPure XP Beads (AXP) by vortexing.
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To the pool of barcoded samples, add a 0.6X volume ratio of resuspended AMPure XP Beads (AXP) and mix by pipetting:
Volume of barcoded sample pool 37.5 μl 75 μl 150 μl 300 μl 600 μl Volume of AMPure XP Beads (AXP) 22.5 μl 45 μl 90 μl 180 μl 360 μl Note: Table contains example volumes for reference. Please adjust the volume of AMPure XP Beads (AXP) added for the volume of your barcoded sample pool to ensure a 0.6X volume ratio.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 2 ml of fresh 80% ethanol in nuclease-free water.
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Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colourless. Keep the tube on the magnetic rack, 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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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). Spin down and incubate for 5 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 10–50 fmol of your eluted samples into a clean 1.5 ml Eppendorf DNA LoBind tube. Make up the volume to 11 µl with Elution Buffer (EB).
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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.