problems with RNAs denatured with Glyoxal(in Northerns)

Reese Bolinger anmab2 at orion.alaska.edu
Wed Feb 28 04:01:46 EST 1996


sb at mole.bio.cam.ac.uk (Saverio Brogna (Genetics)) wrote:
>Please someone,
>
>could you explaine why when I run Northerns with RNAs denetured in Glyoxal
>I see two bands from my transcript, and anly one when I used the more
>commun formaldeyde gels. I dont think that is due to a better resolution
>of the first method, but I rather think that is an artifact.
>Do you have any suggestions.
>
>Thanks Saverio

Hi:

We've run into similar problems when using glyoxal gels for total RNA northerns in the past.  You didn't mention how much RNA you were loading onto the gel or the conditions under which you were treating it, but it's possible to "overload" the glyoxal/DMSO mixture with RNA, resulting in incomplete denaturation.  Treating the RNA in a larger volume may overcame your problem.  The following mixture is what I use for a "routine" glyoxalization reaction:

1.6 ul 0.10 M NaH2PO4,  pH 7.0 DEPC treated
2.67 ul 6 M deionized Glyoxal
8 ul DMSO (tissue culture grade)
up to 3.73 ul RNA and nuclease free water (Total Volume = 16 ul)

This works for an upper limit of about 20 ug of total RNA in my experience.  If you haven't tried it, you could stain a gel with acridine orange to verify complete denaturation by the color difference this gives depending on whether the nucleic acid is double or single stranded.  Hope this helps.

                                             Reese






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