acid phenol

Neil Saunders saunders at bio.vu.nl
Tue Dec 8 02:23:33 EST 1998


> does anyone have any experience using acid phenol to isolate RNA from a
> complex mixture that includes genomic DNA?
>
> thanks for any tips
>
> jane


My experience using Gram-negative bacteria has been that there is always
quite substantial genomic DNA contamination using this method.  Admittedly,
I've only tried it a few times, before resorting to a Dnase-treatment step
(which is recommended anyway, for any sensitive applications like RT-PCR,
5'-RACE etc), and including the Dnase isn't really a lot more work.  I
think it's very important to prepare the acidic buffer with care so as to
end up with the right result.  Most papers refer to 2 M sodium acetate pH
4, which is one of those buffers that you have to prepare carefully (it's
2M total acetate, so making 2M sodium acetate and adding acetic acid
doesn't work!).  Appligene also include a buffer concentrate with their
Aquaphenol solution for making acidic phenol (I think at pH 5 or so), again
it's important to allow the solution to equilibrate properly, and I think
it's Gibco who market a Trizol reagent based on the original acid phenol
method of Chomczynski and Sacchi.  It probably also depends to some degree
on the starting material; the majority of papers I've read refer to
eukaryotic cells or tissues, as do most RNA isolation protocols, and they
all seem able to use acidic phenol without much trouble.

Good luck!

Neil Saunders

--
Department of Molecular Cell Physiology,
Faculty of Biology,
Vrije Universiteit,
De Boelelaan 1087,
1081 HV, Amsterdam
The Netherlands

phone: +31 20 4447194
email: saunders at bio.vu.nl
WWW: http://members.xoom.com/paracoccus/





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