- Materials
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- 100-150 ng high molecular weight genomic DNA
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
- Fragmentation Mix (FRA)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler or heat blocks
- P2 pipette and tips
- P10 pipette and tips
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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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Thaw the 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 Fragmentation Mix (FRA) Not frozen ✓ ✓ Rapid Adapter (RA) Not frozen ✓ ✓ Adapter Buffer (ADB) Not frozen ✓ ✓ -
Once thawed, keep all the kit components on ice.
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Prepare the DNA in nuclease-free water.
- Transfer 100-150 ng genomic DNA into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to 10 µl with nuclease-free water
- Mix by flicking the tube to avoid 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 100-150 ng template DNA 10 μl Fragmentation Mix (FRA) 1 μl Total 11 μl -
Mix gently by flicking the tube, and spin down.
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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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The tagmented DNA in 11 μl is taken into the adapter attachment step.
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In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:
Reagents Volume Rapid Adapter (RA) 1.5 μl Adapter Buffer (ADB) 3.5 μl Total 5 μl -
Add 1 μl of diluted Rapid Adapter (RA) to the tagmented 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.