- Materials
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- 10 µg of extracted high molecular weight genomic DNA
- Short Fragment Eliminator (EXP-SFE001) kit
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 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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- Centrifuge
- Ice bucket with ice
- Heating block
- Wide-bore pipette tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
- Agilent Femto Pulse System (or equivalent for read length QC)
- Optional equipment
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- Shaking heat block
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Optional actionWe recommend substituting nuclease-free water for TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) if the library is going to be stored over a long-term period.
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In a 1.5 ml Eppendorf DNA LoBind tube, prepare 10 µg DNA in 100 µl of nuclease-free water to a final concentration of ~100 ng/μl.
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Add an equal volume of the Short Fragment Eliminator (SFE) buffer to the DNA sample and mix thoroughly by gently flicking the tube until homogenous.
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Place the tube in the centrifuge and note the orientation of the tube within the rotor.
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Centrifuge the sample at 10,000 x g at room temperature for 30 minutes.
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Aspirate and discard the supernatant, taking care not to disturb the pellet.
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Without disturbing the pellet, add 200 μl of freshly prepared 70% ethanol to the tube. Centrifuge the sample at 10,000 x g for 3 minutes at the same orientation used in for the previous centrifuge step. Pipette off the ethanol and discard.
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Repeat the previous step.
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Add 50 μl of nuclease-free water to the DNA pellet and mix by gently flicking the tube.
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Incubate the tube in a heat block at 37°C for 30 minutes. Gently agitate the solution by flicking every 5 minutes to aid with resuspension. Alternatively, use an incubated shaking heat block at 37°C, 300 rpm for 30 minutes.
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Gently mix the tube contents by pipetting up and down using a wide-bore tip.
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Quantify the sample three times using the Qubit dsDNA BR Assay Kit, ensuring that replicate Qubit measurements are consistent before continuing to library preparation.
If the Qubit measurements are not consistent, this could indicate that the DNA has not been homogeneously resuspended. If this occurs, we recommend increasing the elution time and incubating the DNA at 50°C to aid with resuspension of the DNA pellet.
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Quantify sample read length using the Agilent Femto Pulse System. An N50 of 30 kb or above is to be expected.