Can anyone suggest which are the critical stages in the comet assay where I must to work in the dark?
I didnt perform any comet assay step in dark, the only fluorescent dye used is EtBr. But I stored the slides in dark though.
Thanks Rahul!! I have always worked all comet assay steps in the dark, but I think that it isn't needed. I think in the electrophoresis step the light could be critical since the naked DNA is unwound. Could the normal light introduce extra DNA damage?
We have been doing Comet assay under yellow light (40W bulb) to avoid possible DNA damage under white light (inderect Sun light or light from Fluoroscent tubes).
We also do the electrophoresis step in the dark or under red light while necessary. I've actually heard a story from an advisor in our lab that when not working in darkness the height of the neon lighting bulbs in the lab has a significant effect on the tail lengths of the negative controls, e.g. cells handled in lower rooms have longer tails. Therefore I would strongly recommend to work under minimal (less destructive) yellow or red light turned on only as long as necessary.
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