RNA gels - staining and denaturing

Andrea Moriondo amoriond at galactica.it
Wed Jul 19 16:19:39 EST 1995


amarion at moose.uvm.edu (Amy Marion) wrote in article 
>
>What is the proper procedure for staining RNA gels (either glyoxal or 
>formamide) with ethidium bromide? 
> 
Hallo,

 I make my formamide-denaturing RNA gels after the references of:

Lenhrach, H. et al. , Biochemistry 16:4743, 1977
Thomas, P., Proc. Natl. Acad. Sci., USA 77:5201, 1980
That is, 1% agarose-0.66M Formaldehyde 37% (in contrast with 2.2M of 
the original procedure, to obtain a better staining with EtBr), and 20 
ul EtBr 10mg/ml for a 300ml gel solution.
Then use 1X MOPS buffer as running buffer and run the gel at 100V for 
at least 1h to obtain a slight separation and check if your RNA is 
still where it has to be, that is, in the gel!
I load the gel with 10ug of total RNA vacuum dried and resuspended in 
20 ul loading buffer, heated to 95°C for 2 min. before loading.

Then I blot the RNA to a Hybond-C filter without any problem. I don't 
know if a Nylon filter can give less result in blotting than my NC 
filter.
Up to date the only problem I have is to find on the blot what I'm 
looking for :-)
Good Luck!

Andrea Moriondo
Universita' degli Studi di Milano
Sede di Varese, via Ravasi, 2
21100 Varese, Italy
e-mail amoriond at galactica.it




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