I am performing comet assay for the first time. My problem is that I have treated normal isolated lymphocytes with 100 uM H2O2 but the cells are not showing any comets. they are not showing any halos either. To me it seems like the cells are not lysed although I have given them lysis time of 24 hours. I haven't added SDS in the lysis buffer only Triton-X 100. Also I haven't used PBS in the agarose solution. Please can anyone tell me where I am doing wrong? ALso please can anyone tell me what is the orientation of slides in the gel tank. Is it horizontal or perpendicular to the electrodes. I am new to CA so any help is greatly appreciated.