I've performed the alkaline comet assay on isolated zebrafish cells, and sometimes I find some nucleoids with this "granular" appearance, without forming a tail, both in negative control and treated cells, at the same time that I find " normal " nucleoids (red box of the image). My positive control is working well, using the same cells, so I excluded that it is a problem with electrophoresis. Before starting electrophoresis, I leave it in fresh lysis solution (2.5M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, 10% DMSO) for 1h30, and in unwinding (300 mM NaOH, 1 mM EDTA ) for 20 min. Has anyone seen nucleoids with this appearance? Any tips?
*attached are some images of these "granular" nucleoids, in red box there is a "normal" nucleoid from the same treatment.
Hey Flavia.
I have done a lot of comet assays and haven't had any of this kind of problem. My first thought was inefficient lysis if I have to guess.
To compare, working with fish samples, I usually let 2 h in a freshly prepared lysis solution [2.5M NaCl, 100mM EDTA, 10mM Tris-HCl, 1% N-Lauryl sarcosine (pH 10)], 1% Triton X-100 and 10% DMSO (the Triton and DMSO added before the use).
I wish you luck.
Sincerely, Rodrigo
- Badges
- Science topic