Improvement of single-cell gel electrophoresis (SCGE) alkaline comet assay

Although the single-cell gel electrophoresis (SCGE) alkaline comet assay can detect DNA damage quickly and with high sensitivity, it does not work well for cryopreserved sperm of some marine teleosts. Using large yellow croaker Pseudosciaena crocea sperm that were cryopreserved in increasing concentrations (5 to 30%) of DMSO, we made modifications to the classic SCGE method that included using common microscopic slides without rough surfaces, pretreatment of sperm before gel spread, and a single layer of gel. Electrophoresis conditions were 130 mA, 15 V, and 60 min, providing a high SCGE sensitivity and definition of the comet image. The improved method is useful for detecting cryopreserved sperm DNA damage of some marine teleosts, especially for testing the quality of genetic resources.