Abstract
The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair.
Keywords: Comet assay, Genotoxicity, DNA damage
Materials and Reagents
Equipment
Software
Procedure
Recipes
Acknowledgments
This protocol was adapted from previously published papers (Courilleau et al., 2012; Olive et al., 1990; Ostling and Johanson, 1984; Ostling and Johanson, 1987; Wojewodzka et al., 2002).
References
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There are different critical steps about the gel (especially the points 3.2 to 3.8 of the on-line protocol) that can influence how it will stick during all the experiment. The first important thing is the temperature of the slide and of the buffers. Everything must be cold. The room temperature must also not be too high. The time (5 to 10 minutes) for which the slide stay on ice-pack and the temperature of the ice-pack is also important (take it out of the freezer 10-20 minutes before the use for the experiment). It is also important to slide off very gently the coverslip after the agarose has solidified.
Thank you so much. The cold temperature tip really helped :)