- Consumables
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- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- 1 M Tris-HCl, pH 7.5
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- 5 M NaCl (Sigma, 71386)
- PEG 8000, 50% w/v (Rigaku Reagents, cat # 25322-68-3)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
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- Magnetic rack
- Hula mixer (gentle rotator mixer)
- Thermal cycler or heat block
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Wide-bore pipette tips
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Custom SPRI bead suspension preparation for Pore-C extraction
Before starting the Pore-C experiment, a custom SPRI bead suspension needs to be prepared. This will be used to deplete non-chimeric monomers and to maximise the frequency of chimeric Pore-C polymers, improving purity ratios and read lengths at the end of Day 3: Pore-C experiment.
We also recommend using this custom SPRI bead suspension to size-select the extracted gDNA input in the Whole blood sample gDNA extraction for the Duplex experiment to enrich for fragments above 1.5-2 kb.
Store the beads at 4°C and bring to room temperature before use.
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Prepare a custom buffer in a 2 ml Eppendorf DNA LoBind tube as follows for use in step 7.
Reagent Final Volume Tris-HCl, 1 M 10 mM 20 μl EDTA, pH 8, 0.5 M 1 mM 4 μl NaCl, 5 M 1.6 M 640 μl PEG 8000, 50% (w/v) 11% (w/v) 440 μl Nuclease-free water - 888 μl Total - 1992 μl Note: We recommend using wide-bore 1 ml pipette tips to accurately pipette 440 μl of 50% PEG 8000.
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Transfer 1 ml of resuspended Agencourt AMPure XP beads into two 2 ml Eppendorf DNA LoBind tubes, so that each tube contains 1 ml.
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Place the tubes on a magnetic rack to pellet the beads until the solution is clear and colourless. Pipette off and discard the supernatant.
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Remove the tubes from the magnet and resuspend the pellets with 1 ml of nuclease-free water. Pellet the beads on the magnet until supernatant is clear and colourless and pipette off the supernatant.
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Repeat the previous step.
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Spin down and place the tubes back on the magnet to pipette off any residual water.
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Resuspend both tubes of pelleted beads in 200 µl of custom buffer and then pool both tubes into a single tube to a total of 400 µl.
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Transfer the remaining custom buffer into the tube containing the pooled beads.