Formaldehyde vs. Formamide for Northern

Kajetan H. Groicher Kajetan-Groicher at ouhsc.edu
Thu Apr 27 10:22:16 EST 2000


in article 3906AAFD.A180D195 at neurochem.u-strasbg.fr, Paul Klosen at
klosen at neurochem.u-strasbg.fr wrote on 4/26/00 3:38 AM:

> Vellanoweth's Lab wrote:
>> 
>> We've been having a lot of trouble degrading our plant RNA with
>> formaldehyde.  We've tried pHing the formaldehyde with NaOH to 5.3, and
>> eliminating the formaldehyde from the incubation step.  However we are
>> still getting degradation in the gel.  I looked in the Ambion catalog to
>> check if our formaldehyde was bio-reagent quality and found they don't
>> sell formaldehyde, only formamide.  This leads me to wonder whether
>> formamide can be used in a denaturing gel rather than formaldehyde, or
>> if you can run it without the denaturant since the sample is already in
>> formamide.  Any northern experts out there?
> 
> Formaldehyde is a potent inactivator of almost all enzymes, because it
> reacts covalently with proteins. It even inactivates RNAses. If you have
> trouble with degraded RNA in your gels, the RNA probably is already
> degraded before it got into your gel. Are you certain about the
> integrity of your RNA ?? Maybe you are dealing with incomplete
> denaturation of you RNA before electrophoresis ??


Incomplete denaturation may be the problem.  I make a dilution buffer with
deionoized formamide, then incubate the RNA at 55oC for 15 min prior to
addition of loading buffer and electrophoresis.  I have on occasion checked
the quality/integrity of my RNA on a standard TAE agarose gel (i.e. no
special RNase protection or buffers etc, just a plain old TAE gel.) and have
consistently seen only the rRNA bands with no apparent degradation.

I am not familiar with a formamide containing gel.  However if you don't
mind the toxicity and need for recirculation you could try a glyoxal gel.

So I guess my suggestions would be, use a formamide buffer to dilute and
denature your RNA in, you may be seeing a simple problem of incomplete
denaturation.  And make sure your RNA is good high quality stuff.

I





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