Q: What damage can be detected with this assay?
A: The Comet assay gives a global picture of DNA damage because it can measure changes caused by double strand breaks, single strand breaks and AP sites. The DNA damage that is able to be detected depends on the running buffer that is selected for the assay. TBE buffer will allow detection of single stranded and double stranded DNA breaks and possibly a few AP sites. An alkaline buffer will detect those as well as AP sites and alkali labile DNA adducts.
Q: What is the limit of detection with this assay?
A: The Comet assay is sensitive to about 50 breaks per diploid mammalian cell; however the results are typically presented as tail moment and tail DNA% (see page 8 of the product manual). The DNA damage that is able to be detected will depend on the running buffer selected for the assay. Unlike an ELISA, the Comet assay does not provide a control because the results of any Comet assay will be variable depending on the experimental conditions. For example, cells containing the same amount of DNA damage may have different tail sizes if the electrophoresis running conditions are different.
Q: Does the entire Comet Slide need to be used all at once?
A: The Comet Slide has a treated surface and once the entire slide is subjected to the protocol steps of denaturation, electrophoresis, and washing it can’t be used again. The 96-well Comet Slide was designed to be used entirely at one time.
Q: How can I prevent the agarose gel from falling off the slide?
A: The Comet slides are chemically treated with an adhesive surface that is designed to hold the agarose in place. Problems with the gel sliding off are due to incomplete coverage when applying the agarose-cell suspension to the slide. The gel must be spread out to cover the entire well. To make sure the agarose gel is firmly attached to the Comet slide, step 4 on page 6 of the product manual is very critical:
Combine cell samples with Comet Agarose at 1:10 ratio (v/v), titrate to mix, and immediately pipette 75 µL/well onto the OxiSelect™ Comet Slide. Ensure complete well coverage by spreading the suspension over the well with the pipette tip.
It is also important that the slide is maintained horizontally the entire time.
A: No, you cannot use a regular microscopy slide for the Comet Assay. Our Comet slides have been pretreated chemically to increase adhesion. Your cell/agarose droplet will not be able to stay on a regular slide.
Q: How can damage to control cells be minimized?
A: The comet assay, also known as single cell gel electrophoresis (SCGE) assay, measures DNA damage of single cell suspensions. All cells will show some DNA damage, and the amount detected may be endogenous levels. In the case of post-harvest damage, you will want to make sure you isolate both cell populations at the same time and under the same conditions. It is also recommended to follow the sample preparation outlined on page 6 of the manual, which is designed to minimize DNA damage due to sample preparation. The assay conditions can also be optimized by running the electrophoresis for a shorter amount of time, which should produce shorter tails with the control cells while maintaining the tails of the treated cells.
Q: What is a good positive control?
A: We treat cells with 20uM etoposide for 4 hours to create a comet tail. We also offer treated and untreated Comet Assay Control Cells.
Q: Is it necessary to perform the experiment under low light conditions once the cells are in agarose on the slide?
A: We recommend covering samples to minimize UV light exposure, which can induce additional DNA damage and result in high background. This is important when preparing your samples but it is probably fine to run the electrophoresis uncovered. The Vista Green dye should also be protected from light because it is light sensitive.
Q: How do I adjust the current to maintain 200mA?
A: 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. In our experience, not much buffer is required to maintain this current.
Q: How long can the slides be stored in Vista Green dye?
A: Once the slides are stained with Vista Green, they can be stored for 3 months in the dark at 4ºC without the fluorescence fading. We recommend staining the slide on the same day that you will be analyzing the results. Slides that are dried and not stained with Vista Green can be stored for one year.
Q: Do you have reference values for DNA damage inducing compounds used for this assay?
A: Unlike an ELISA, the Comet Assay does not provide a control because the results of any Comet Assay will be variable depending on the experimental conditions. For example, cells containing the same amount of DNA damage may have different tail sizes if the electrophoresis running conditions are different. Comparisons to our results or results from another researcher will be meaningless because the conditions will never be the same between the two. If you are interested in testing the effects of compounds on DNA damage, our recommendation is to keep all conditions consistent between experiments and use the same running time and software for analysis.