I am detecting digested, P32 terminally labelled RNA using large polyacrylamide gel. I prerun the gel for 1 hr to make sure it's hot and run my gel for ~2 hrs in 1x TBE buffer at 1600V. I expose the film in a cassette with intensifier screen at -80C. My picture comes out blurry most of the time, or not evenly sharp. What can be the most likely cause of blurriness and is there a way to make the bends sharper?
I think u can fix the gel in 20-30% methanol..this will give u sharper bands..
As Matthias said, it is condensation that causes the blurriness. To avoid this, take the cassette out of the -80C and leave it at RT for about 30min before developing the film.
Sorry, I forgot to add: If you have a plastic sheet between the gel and the film, it might also cause blurriness, but this would be even, not patchy. The best option is, as suggested above, to dry the gel on a piece of whatman paper etc so the sample does not diffuse o/n, then wrap the dried gel in glad wrap (because this is really thin) and then put the film on.
Thanks Sandra. Thats normally what I do, I dry my gel for 1 hour in gel drier on watman paper, wrapped in plastic wrap and then expose. I think polymerizing the gel for more than 1 hour and exposing at room temp. might do the trick. Thank you everyone for help!
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