- Materials
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- Custom SPRI bead suspension
- Consumables
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- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Chilled phenol:chloroform:isoamyl alcohol in a 25:24:1 ratio, saturated with 10 mM Tris.HCl pH 8.0, 1 mM EDTA (Sigma, P3803-400ML)
- 5 M NaCl (Sigma, 71386)
- 3 M sodium acetate, pH 5.5 (Invitrogen, AM9740)
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- Ethanol, 100% (e.g. Fisher, 16606002)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 5 ml centrifuge tubes
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
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- Temperature-controlled centrifuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Thermal cycler or heat block
- Qubit fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- -80°C freezer storage
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Day 3: Pore-C experiment overview
On the third day of the Pore-C experiment, the chimeric Pore-C dsDNA polymers are purified from the solution of polypeptide fragments and residual reaction buffers. The peptides are removed by using a phenol:chloroform extraction, followed by an ethanol precipitation to purify the DNA from the residual reaction buffers and phenol. The final Pore-C DNA extract is a pool of chimeric dsDNA polymers made of multiple ligated monomers which are sequenced to determine DNA interactions, proximity in sequence space and the three-dimensional structures of chromatin within the nucleus.
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Pre-cool the centrifuge to 15°C.
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Place the sample on ice until cool, then transfer the entire volume to a 5 ml centrifuge tube.
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Rinse the original tube with a further 200 μl of nuclease-free water and add this to the same 5 ml centrifuge tube for a total sample volume of ~2200 μl.
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Add an equal volume of chilled phenol:chloroform:isoamyl alcohol 25:24:1 saturated with 10 mM Tris.HCl pH 8.0, 1 mM EDTA, adjusting this volume as needed to match that of the sample. Mix by gently inverting the tube for 5 minutes to achieve a homogeneous emulsion.
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Centrifuge the aliquots at 16,000 x g at 15°C for 15 minutes.
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Incubate the aliquots on ice for 2 minutes until the organic phase becomes cloudy; this will strengthen the integrity of the interphase layer.
If the protein degradation has been successful, the interphase layer will be very thin and clear.
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Transfer the aqueous phase into a fresh 5 ml centrifuge tube for each aliquot and make note of the recovered volume (expect ~2,000 μl).
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Transfer half of the recovered aqueous phase to a second 5 ml centrifuge tube to create two equal aliquots.
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For each aliquot, add 0.02X of 5 M NaCl (0.1 M final) and 0.1X of 3 M sodium acetate pH 5.5 (0.3 M final), relative to the volume of the recovered aqueous phase of the aliquot. Mix by gently inverting the tube.
The solution will likely turn cloudy and then become clear once again.
For example, for a recovered volume of 2,000 µl, add:
- 40 µl of 5 M NaCl
- 200 µl of 3 M sodium acetate
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For each aliquot, add 3X of 100% ethanol relative to the volume of the recovered aqueous phase. Mix by gently inverting the tubes.
The solution will likely turn cloudy and then become clear once again.
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Precipitate at –80°C for >1 hour.
Note: If a –80°C freezer is not available or a pause in the protocol is required, an overnight incubation at –20°C can be used instead.
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Pre-cool a centrifuge to 4°C.
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Centrifuge the sample at 16,000 x g at 4°C for 30 minutes.
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Aspirate and discard the supernatant, then wash the pellets with 4 ml of 80% ethanol.
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Centrifuge the sample at 16,000 x g at 4°C for 5 minutes.
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Aspirate and discard the supernatant, then wash the pellets with 2 ml of 70% ethanol.
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Centrifuge the sample at 16,000 x g at 4°C for 5 minutes.
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Aspirate and discard the supernatant. Briefly spin down the tubes and aspirate any residual supernatant. Allow the pellets to dry for 5 minutes.
After the DNA pellets have dried, they may loosen from the tube.
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Carefully resuspend each aliquot in 75 μl of TE buffer. Incubate for 5 minutes at room temperature, mixing by gently inverting the tube every few minutes.
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Briefly spin down the tubes, then transfer and pool all aliquots together into a 1.5 ml Eppendorf DNA LoBind tube.
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Quantify DNA concentration by using the Qubit dsDNA HS Assay Kit. Ensure a 1/10 dilution is used, as the Qubit reading will be affected by high salt concentration.
Note: The expected yield is ~7 μg per 10 million cells input for cell culture. Yields for other sample types may be reduced.
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Dilute your sample to 60 ng/µl in a final volume of 50 µl of TE buffer at pH 8.
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Add 42.5 µl (0.85X) of room temperature custom SPRI bead suspension and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Spin down briefly and pellet on a magnet until the supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the tube from the magnetic rack and resuspend the pellet in 40 µl of TE buffer. Incubate for 1 minute at 50°C, and then for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
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Remove and retain 40 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of eluted sample using a Qubit fluorometer.
You can expect a 50-55% loss of DNA depending on a fragment length distribution of input material: the greater the proportion of short fragments (<1.5-2 kb), the greater the sample loss.