I’m planning to run the Comet Assay to assess DNA damage, but I’m debating whether it’s worth investing in a specialized electrophoresis tank or if a standard tank can handle it well enough. Has anyone tried both? I’d love to hear any tips or experiences on whether the specialized tank really makes a difference in terms of accuracy or reproducibility.
Hello Seda Nur Homurlu
Standard tank will suffice. It's not worth investing in a specialized electrophoresis tank. You just have to incorporate a few points in your protocol.
During the electrophoresis step of comet assay, the high current can cause several problems.
High current can generate heat which can cause an increase in temperature that can denature DNA or cause smearing of DNA on the gel. It can also cause uneven migration of DNA fragments. To overcome this problem, specialized electrophoresis tanks for the Comet Assay are equipped with connections for a recirculating water chiller to keep the tank at a constant temperature during electrophoresis and eliminate joule heating effects. These tanks can be used with ethylene glycol, allowing cooling to 4°C in the electrophoresis buffer. Also, they are constructed from opaque acrylic, so that no light can penetrate the tank during electrophoresis.
Without using these specialized tanks, you too can get rid of these problems. Do the following.
1. After the lysis step, carefully remove the slide carrier from the dish. Gently place the slide carrier in a washing dish pre-loaded with ice-cold double distilled H2O and leave it for 30 min ensuring that slides are completely covered with double distilled H2O.
2. Insert a frozen cooling pack inside the sliding drawer under the electrophoresis tank to maintain optimal buffer temperature.
3. Carefully add ice-cold electrophoresis working solution to the electrophoresis tank and transfer the slide carrier into the electrophoresis tank.
4. Allow the slides to sit in the electrophoresis tank for 20 min so that the DNA relaxes and unwinds. Keep the power supply switched off during this step.
5. If needed, insert a new frozen cooling pack to maximize chilling.
6. Maintaining the current will be influenced by the buffer volume in the electrophoresis tank. Instead of filling the tank as you would if running a gel, the buffer level should start by just barely covering the slide. Adjustments to the level can then be made until a current can be maintained.
7. Perform electrophoresis for 20 min at 1.19 V/cm, or whatever conditions you may have optimized for the run.
Another point which I would like to mention is that covering samples to minimize UV light exposure is necessary, which can induce additional DNA damage and result in high background. This would be important when preparing your samples, but it is probably fine to run the electrophoresis uncovered.
Best.
Hi Nobre,
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