Hi all,
I have been trying to establish this technique in lab, but so far, I can't get a clear result. The protocol I am following is this:
https://www.jove.com/video/3255/visualization-dna-replication-vertebrate-model-system-dt40-using-dna
When I spread the DNA, I see the opaque line they describe. However, I can't see the fibers under the microscope. The problem I have is that I don't see the clear fibers as they do. I counterstain the DNA with DAPI at the end of the experiment, and I can't see any fibers like the ones from the protocol. In fact, when I find something similar to the fibers, it is stained with the three colors, what makes me think that it's not truly DNA but rather some sort of scratch or artifact.
Does anyone have ever encountered a similar problem? Do you have another protocol I could follow? What magnification do you usually use (I use 20X, which I believe should be enough)? I attach a picture of the "fibers" I see, it's not the sharpest (and has a high background).
Thank you for your advice!
Hi!
Are you sure your cells are incorporating the analogs? Maybe you should double check that.
Anyway I always use higher magnification like 40X or 63X (fibers can be really small).
Hope this helps!
Hi! Did you solve your problem?
I also followed this protocol in jove, and I can see the labeling DNA fibers.
Maybe there are something you could notion.
You'd better use the agents and antibodies from the same company as described in this protocol.
IdU is not easy to resolve. It requires more than an hour of agitation at 37°C in a shaker. You should make sure IdU is completely resolved before you add it into cell culture.
I can see the fibers when I use 40X objective lens, and clearer fibers will be seen with 100X objective lens.
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