- Consumables
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- T4 DNA Ligase 400,000 U/ml (NEB, M0202S/L)
- Recombinant Albumin at 20 μg/μl (NEB, B9200S)
- Tween-20 (Thermo Scientific, J20605.AP)
- Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
- Proteinase K at 20 μg/μl (NEB, P8107S)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
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- Thermomixer
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
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Day 2: Pore-C experiment overview
For Day 2, after the overnight incubation, the restriction enzymes are heat inactivated to prevent re-digesting ligated products. DNA ligase is added to the clusters of crosslinked DNA and passively diffuses through the crosslinked cytoskeleton cage to ligate the cohesive ends of proximal monomers into chimeric Pore-C polymers. After ligation, the ligated products can be released from the crosslinked cytoskeleton cages by an overnight proteinase K digestion. This releases the chimeric Pore-C polymers into solution as dsDNA.
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Thaw the T4 DNA Ligase and T4 DNA Ligase Reaction Buffer in accordance with the manufacturer's instructions and place on ice.
- Thaw the reagents on ice.
- Flick and/or invert the reagent tube(s) to ensure they are well mixed.
Note: Do not vortex the T4 DNA Ligase enzyme. - Spin down tubes before opening for the first time each day.
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Prepare 5 ml of 20% Tween-20 in nuclease free water as follows:
- Weigh out 1.095 g of Tween-20 and transfer to a fresh 5 ml centrifuge tube.
- Add 4 ml of nuclease-free water.
- Gently invert the tube until the solution is homogenous.
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Set the thermomixer to 65°C.
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Heat denature the restriction enzyme by incubating the sample suspension in the thermomixer at 65°C with 300 rpm rotation for 20 minutes. Allow the reaction to cool to room temperature.
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Set the thermomixer to 16°C.
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Set up the proximity ligation reaction according to the table below, adding reagents directly to the sample suspension in the following order. Mix gently by pipetting up and down, using a wide-bore pipette tip.
Reagent Final Volume Digestion reaction (from Day 1) - 450 µl Nuclease-free water - 395 µl T4 DNA Ligase Reaction Buffer, 10X 1X 100 µl Recombinant albumin, 20 µg/µl 0.1 µg/µl 5 µl T4 DNA Ligase, 400 U/µl 20 U/µl 50 µl Total - 1000 µl -
Incubate the sample suspension in a thermomixer at 16°C for 6 hours, with periodic <1000 RPM rotation for <30 seconds every 15 minutes. This prevents condensation inside the lid.
Note: This incubation can be performed without mixing.
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Set the thermomixer to 56°C.
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Add the reagents to the previous ligation reaction in the following order to make up the protein degradation reaction. Mix the sample gently by inverting the tube 3–4 times.
Reagent Final Volume Ligation reaction (from the Proximity Ligation) - 1000 μl Nuclease-free water - 300 μl Tween-20, 20% 5% 500 μl SDS, 10% 0.5% 100 μl Proteinase K, 20 μg/μl 1 μg/μl 100 μl Total - 2000 μl -
Incubate the sample suspension in a thermomixer at 56°C for 18 hours with periodic <1000 rpm rotation for <30 seconds every 15 minutes to prevent condensation inside the lid.
Note: This incubation can be performed without mixing.