peterg at rnaworld.bio.ukans.edu
Mon Dec 19 18:31:59 EST 1994
In <hrasal.223.000BAB94 at marc.cri.nz>, hrasal at marc.cri.nz writes:
>There are two things that I'm curious about. When I first started
>working with RNA, I was told that autoclaving inactivated RNAses, but the
>RNAse protein will (after a certain period, 24 hours or so), reform into the
>active enzyme. I had assumed that this was stated in Stryers Biochemistry. I
>was wrong. Actually, it was denaturation of ribonuclease in 6M guanidine HCl
>followed by removal of the guanidine by dialysis. So, is reactivation of
>RNAse activity after autoclaving an urban legend, or is it real?
** It is, like all legends, based on a grain of fact. The structure of RNase A
is held together by disulfide bonds, so if it is autoclaved and then SLOWLY
cooled, it may renature. (This is why many RNA isolation protocols include high
concentrations of BME.) In practice, most RNA labs I know find that autoclaving
gives buffers which are RNase-free. Glassware should be oven-baked overnight
for best effect, but autoclaving of dry (=fast cool) cycle works.
>Also, phenol is used in the RNA extraction protocol that I use. The phenol is
>equilibrated with Tris-HCl as prescribed by Sambrook, Fritsch and Maniatis'
>Molecular Cloning. In it, they state that the phenol should be equilibrated
>to pH >7.8 as DNA (and I assume RNA) partitions into the organic phase at acid
** Linear DNA is soluble in phenol at acid pH (<= pH 5). RNA and plasmid DNA are
not; they stay in the aqueous phase. This fact has been used to separate RNA and
DNA for at least 30 years. It has just been "rediscovered" as the basis of
several commercial kits. The reason phenol should be equilibrated with the same
buffer used for the aqueous phase is that liquid phenol is 88% phenol and 12%
water, so when you do a phenol extraction, the aqueous component in the phenol
mixes with your aqueous solution of nucleic acid. If you want close control over
buffer components and pH, pre-equilibration of the phenol is a must.
Now, RNA will hydrolyse under alkaline conditions. My second question
>is this: At what pH and/or concentration of alkali does RNA hydrolyse? I've
>checked Sambrook, Stryer and the Merck index, unsuccessfully. FYI, I use
>phenol equilibrated to pH 8.0. For Northerns, I blot my RNA onto HybondN+
>with 50 mM NaOH.
** Other posters have good answers to this. OH- mediated hydrolysis of RNA is
dependent on [OH-] and temperature. At 0-deg-C and pH 8.0, RNA is pretty
stable. At 100 degrees C, RNA will be hydrolyzed in unbuffered water. Inclusion
of EDTA (e.g., TE buffer) will chelate divalent cations which promote formation
of OH radical and OH-.
| Peter Gegenheimer | pgegen at kuhub.cc.ukans.edu |
| Departments of Biochemistry | voice: 913-864-3939 |
| and of Botany | |
| University of Kansas | FAX : 913-864-5321 |
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