- Materials
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- 2 µg of extracted gDNA (from previous step)
- Consumables
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- g-TUBE™ (Covaris, Cat. # 520079)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Eppendorf 5424 centrifuge (or equivalent)
- 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)
- Optional equipment
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- Agilent Femto Pulse System (or equivalent for read length QC)
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Fragmentation of extracted DNA using Covaris g-Tube:
To prepare fragmented gDNA for the library prep protocol, mechanical fragmentation is performed using a g-TUBE (Covaris) to shear DNA to a fragment length of approximately 6kb.
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Prepare the DNA in TE buffer:
- Ensure you have 2 µg of extracted gDNA from the sample extraction, and transfer this into a 1.5 ml Eppendorf tube.
- Adjust the volume to 50 μl with TE buffer.
- Mix thoroughly by pipetting up and down.
- Spin down briefly in a microfuge.
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Load the 50 µl of the sample into the top of the g-TUBE. Screw the cap firmly and centrifuge at 11,000 rpm (~11,300 RCF) for 30 seconds.
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After centrifugation, spin the tube again at 11,000 rpm (~11,300 RCF) for 10 seconds to ensure complete passage of all gDNA through the constriction.
Visually inspect to confirm the entire sample has passed through the upper chamber to the lower chamber of the g-TUBE.
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Invert the g-TUBE and spin it again at the same speed and duration as above: 11,000rpm (~11,300 RCF) for 30 seconds.
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Repeat the centrifugation at 11,000 rpm (~11,300 RCF) for 10 seconds to ensure thorough passage of all gDNA through the constriction.
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Unscrew the tube body, leaving the screw-cap containing the sample. Retrieve the sample from the g-TUBE screw-cap and transfer it into a clean 1.5 ml Eppendof tube.
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Quantify 1 µl of the fragmented gDNA using the Qubit dsDNA Broad Range Assay Kit.
Sample concentration after g-TUBE shearing, is expected to be within the range of 25–35 ng/µl.
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Optional actionThe fragmented gDNA should also be assessed using Femto-Pulse (Agilent) to evaluate the size and quality of the DNA.
Example DNA fragment distribution after g-tube fragmentation, analysed using an Agilent 165 kb Femto-Pulse Assay. Note the single prominent peak ~6 kb.