I have been trying to develop DNA fragmentation by hydrogen peroxide (1mM Concentration). till now i have tried conventional, spin column and DMSO-TE-SDS methods for DNA isolation. But i am not able to get the DNA fragmentation. I am working on HEK293t and C2C12 cell lines. I am collecting cells at different time periods after treatment with hydrogen peroxide (2hr, 4hr, 6hr, 8hr and 24hr).
Any suggestions which can help my research work.
Thanking you in advance.
Hi,
true DNA fragmentation is quite tricky to observe. According to me it is a late event in the PCD process, do you have any cell death kinetic information ? (IP, LDH...). My suggestions would be: try different concentrations of agarose (if you're running southern blot of course) because low low size DNA frgaments may be difficult to see in an underconcentrated agarose gel. Be sure that you get an homogenous dead cell population, for that purpose you should try to mesure DNA fragmentation at later stage of cell death: 48 and 72h maybe. Did you try the TUNEL assay ?
Hi Sir,
Are you want evaluated PCD? If yes, I think innovative techniques to determined this are TUNEL and ISEL assay. These methods are very accurate to DNA fragmentation assay. Also I suggested, you can utilized MetaPhor gel.
Be successful,
T.Roshani
Actually i might have done TUNEL. but it is a costly assay and i need to standardize the positive control without expending too much. I will surely go for TUNEL for my critical samples. This is a positive control which is very well reported but still i am not able to reproduce the results. Thanks for the suggestions. Thanking you metaphor gel information. I was completely unaware of this product. Thank you again T.Roshani..
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