I was doing a comet assay using a Comet assay kit. When I try to visualize DNA of cells under the microscope, I realized the cells are not present in one plane so I could not focus all present in one frame. The cell which were not in focus, appeared as big comets. Can I consider such a cell which was not in focus? How do I fix this problem, or how do I run gel in such a way that all cells are in the same plane (hence all cells are in focus at the same time)?
Dear Ajit,
I think the reason of it is a thick layer agarose gel (containing cells). We've had similar problem in our laboratory and we solved it by decreasing of the volume of agarose for cells. In another way, you can try to spread the agarose with cells on slide to obtain a thinner layer of agarose.
Sincerely,
Maxim
Dear Ajit,
I think the reason of it is a thick layer agarose gel (containing cells). We've had similar problem in our laboratory and we solved it by decreasing of the volume of agarose for cells. In another way, you can try to spread the agarose with cells on slide to obtain a thinner layer of agarose.
Sincerely,
Maxim
Make sure you are using just enough agarose to cover your slide. As Maxim points out, the more agarose used, the thicker the gel will be. Also make sure you are using an appropriate cell density, where you don't have too little or too many cells.
Thanks MAxim and Anne for your answer. I used the volume of agarose recommended by the kit provider. I will certainly try using less volume of agarose. Could you please suggest me some free available softwere to analyse Comet... thanks
I can recommend you CASP software. You can download on this link http://casplab.com/download. It is enough simple to use and it allows to get correct results.
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