Hi Colm Dillon,

Firstly, I would say that you might have done some mistake during the experiment (particularly during extraction, lysis or staining). Secondly, it is not easy to get good comets specially for first time attempters. Please confirm the experimental material and your procedure first and then try to repeat your experiments several times, if you really want to get good ones...

Wish you all the best!

Thanks Muhammad 

I agree with everything Muhammad says! Additionally in order to analyze your putative mistake, you might try to check after every step, whether you lost your cells. Even before the dying procedure you can take a small aliquot after each step and make sure there´s still cells present under the microscope. The fragments you see might still be cell debris, especially if you used too hot agarose or too high of incubation temperature (In case you included any incubation steps in your protocol).

I also agree with all of you. One of the most critical steps that may cause loss of cells is a high temperature of LMP agarose. It has to be at the temperature of 37 degrees. After the step of putting slides into lysis buffer, you can also check cell mixture in agarose in optical microscope. I do this always when can not see any pelet. Good Luck!

Hi Colm Dillon,

Unfortunately, you didn't anything write about conditions of electrophoresis ! It will be useful for any comments to show microphotography of your slides.

Hello Nikolay,

Regarding the conditions, Slides were run for 20 minutes at 26 volts and 300 mA at 4 degrees. I am using specifically prepared slides for comet Assay from Trevigen. I saw lots of debris under the microscope after I had stained.