I am running a neutral comet assay on HS-5 cells and I am having a rather difficult time producing comets. My thought is that the cells aren’t lysing properly. The only time I’ve seen this assay work as intended for the positive control is when my PI left the cells in lysis for 3 weeks which is excessive. Using the same buffers and equipment as him, I left slides in lysis for 35 mins (what the protocol calls for), 1hr, and 24hrs. None of the conditions produced comets. The only real differences between my procedure and his is spin down time (He used 7 minutes and I used 5) and he used EDTA to detach cells while I used trypsin. Would either of these cause an issue with producing comets? Should it take days to lysis these cells?
Here are the buffer solutions:
Lysis Buffer - 2.5M NaCl, 100mM EDTA, 10mM Tris pH 8.8, with final pH 10
Electrophoresis Buffer - 89 mM Tris Base, 89mM Boric acid, and 2.5 mM EDTA disodium salt
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