Hello everybody, I want to try the comet assay, which is new for me.
in order to see whether my treatments induce directly DNA strand breaks or via ROS formation I'd compare samples run with the traditional comet protocol with the one where you induce excisions at sites that are modified due to ROS.
I heard it is recommended to use slights, chamber and lysis solution original from Trevigen. But what with the enzymes that induce excision ?? Does anyone know an alternative/did test it ?
Thanks for any help/tip,
Vanessa
Hi vanessa,
Sorry, I don't know about using special enzyme to investigate genotoxicity from ROS. But for saving cost, you can use a classic chamber (I use a biorad one), and do your lysis buffer home made (the recipes are well described in litterature, including my paper). I agree, slide Trevigen are the best.
Good luck
No I never tried comet assay with aparatus or lysis buffer from Trevigen. I compared my data with my control cells and I always add a positive control (cells treated 5min under UV light from biological hood). The Comet obtained look like the one found in the literrature.
My paper: Effects of doxorubicin and vincristine on undifferentiated rat spermatogonia, Hermance BEAUD Ans van PELT and Geraldine Delbes
Recipe of the lysis solution :2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, and 1% Triton X-100; final pH 10
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