- Materials
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- 2 µg of extracted high molecular weight gDNA
- Consumables
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- g-TUBE™ (Covaris, Cat. # 520079)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- (Optional) TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
- Equipment
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- Microfuge
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Agilent Femto Pulse System (or equivalent for read length QC)
- Qubit fluorometer (or equivalent for QC check)
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Optional actionWe recommend using TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) rather than nuclease-free water if the library is going to be stored over a long-term period.
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Transfer 2 μg extracted gDNA into a 1.5 ml Eppendorf DNA LoBind tube, and adjust the volume to 100 μl with nuclease-free water.
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Mix the DNA thoroughly by flicking the tube. Spin down briefly in a microfuge.
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Transfer the genomic DNA sample in 100 μl to a Covaris g-TUBE™.
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For a fragment length of 10 kb, centrifuge the g-TUBE™ at 4300 x g for one minute at room temperature. Remove and check that all the DNA has passed through the tube.
Note: Please ensure that you are using the correct speed for your equipment and input sample to achieve shearing of 10 kb fragment lengths.
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Invert the g-TUBE™ and centrifuge again for one minute to collect the fragmented DNA. Remove and check that all the DNA has passed through the tube.
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Transfer the 100 μl fragmented DNA into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of eluted sample using a Qubit fluorometer and check sample read length using the Agilent Femto Pulse System. An N50 of 10 kb read lengths is to be expected.