Formaldehyde pH for Northerns?
Gys de Jongh
GysdeJongh at compuserve.com
Wed Apr 5 16:59:52 EST 2000
"Vellanoweth's Lab" <ddeloss at calstatela.edu> wrote in message
news:38EAAFA1.9D679AA1 at calstatela.edu...
> We've been having trouble with our RNA degrading during Northern's.
> We've discovered the culprit is the formaldehyde and particularly the
> denaturing step (we denatured with formaldehyde in the sample). The pH
> of our formaldehyde was (4.4) which we had pH'd from protocols which
> mentioned the pH should be >4. However, after running some analytical
> gels we discovered that a pH of 5.6 or even 6.6 gives much better
> retention of the sample and that we're degrading quite a bit at pH 4.4.
> Basically, I'm wondering if this is a common problem and whether labs pH
> the formaldehyde every time they run a northern, and if so, what is the
> optimum pH?
a solution of formaldehyde gas in water should have a neutral pH. So the optimum
pH=7 , which can not be measured with a pH-electrode because the resistance of
the solution is too high. (There are practically no ions present) The
formaldehyde will be converted to formic acid by air oxigen. Now the,acid, pH
can be measured with a pH electrode. I don't think that neutralising the formic
acid is a good idea. The best thing to do in practice is 1) take a fresh bottle
of formalin ; should last for month if kept tight. 2) make fresh ,non-oxydised
formaldehyde, from para formaldehyde , a polymer of formaldehyde. Imo a very
nice alternative for the formaldehyde is the , reversible, chemical reaction of
RNA with glyoxal in DMSO containing buffer. It forms rings with RNA preventing
H-bonds by sterical hinderence. This reaction product is stabel. So , after the
reaction , the electrophoresis can be done on the bench in a standard buffer.
After blotting to nylon the reaction can be reversed by heating the nylon blot
at 65 pH=8 (tris/cl) for 10 min. See maniatis for details.
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