Hi everyone,
I'm trying to optimise a comet assay for radiation induced DNA damage. I'm following a nature protocol (NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 23) for my procedure. We have bright-field images that show that we have successfully lysed the cells and the the fluorescence images (PI stained) show a good cell density in the gels. My negative control has had no treatment while the positive has been irradiated with 10 Gy (on ice to prevent re-formation of the DNA helices). However, my comets haven't formed for the positive control and look identical to the negative control. All of my protocol was carried out in the dark at 4 oC.
My electrophoresis settings were 25 V, 300 mA, 300 W at constant current for 40 mins in alkali buffer. This is the part that I am most confused with. Suggestions are that I need to run either higher voltage (70-100 V) or maintain the voltage and increase the time? I also have questions about other factors too. Protocols say that a field of 1 V/cm should be used. Do I use the length of my gel or the length between the anode and the cathode? Do I need to prepare fresh run buffer every time or can It be stored in the fridge?
The used proticol is completely accurate. You should check the ph of all of buffers and make them fresh.
Anda Huna thank you for your response. I have bright-field micrographs of the slides and the cells are clearly denatured (cells were lysed overnight in high base and salt).
Is there anything else that you can suggest?
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