Storage of RNA
peterg at rnaworld.bio.ukans.edu
Tue Oct 18 20:56:10 EST 1994
In <37k1mp$jet at canopus.cc.umanitoba.ca>, hamel at cc.umanitoba.ca (Andre Hamel) writes:
>RNA should store well in sterile dH2O in freezer.
>We have MANY aliquots of total RNAs obtained from several types of
>specimens (feces, tissues, whole blood, etc, etc) ... ALL store WELL in
>-20*C PLUS used daily (frozen-thawed MANY times) ... used for RT-PCR ...
>ALL are still okay, right down to last bit used (most aliquots are 50 uL,
>with 1 uL used per reaction).
>If stored in formamide (such as used for northerns) ... simply ppt with
>alcohol and NaAc ... add NaAc to 0.3 M final conc and use 1-2 vol of
>isoprop or 2-3 vol of EtOH, mix well, incubate -20*C for at least 1 hr
>before harvesting pellet ... formamide will be washed away (remains in
>supernatant along with the alcohol).
>Andre Hamel email: hamel at cc.umanitoba.ca
>Manitoba Government tel: 204/945-7630
>Department of Agriculture FAX: 204/945-8062
>Veterinary Services Branch
>Winnipeg, Manitoba, CANADA
>In article <sticknbd-121094224623 at 188.8.131.52> sticknbd at miranda.cc.vanderbilt.edu (B Stickney) writes:
>>In article <1994Oct7.192254.1 at molbiol.ox.ac.uk>, rhubner at molbiol.ox.ac.uk
>> > In article <ros.2.2E911FE4 at molbiol.uct.ac.za>, ros at molbiol.uct.ac.za
>--- stuff deleted ---
>> > > I have been storing RNA samples from both E.coli and Thiobacillus
>>> > ferrooxidans in DEPC water or RNA storage buffer, 20mM sodium phosphate pH
>>> > 6.5 1mM EDTA. The T.ferrooxidans appears to be ok on a agarose gel, but no
>>> > longer gives good results on a Northern Blot, and I presume this is due to
>>> > degradation of the RNA. I store the RNA at -70 C and thaw and refreeze it
>>> > every time I use it, so this could also be causing problems. I see some
>>> > people store their RNA in formamide at -20 C, how do they then get rid of
>>> > the formamide, when they want to do primer extension for example?
There are two preferred ways to store RNA (take it from an old-timer).
1) Especially for smaller RNAs (< a few hundred nt), storage at -20 or -70 degrees in
TE buffer or water works well. There will be absolutely NO degradation IF the RNA
itself is 100% pure (proteinase K-treated, etc, etc), all buffers and storage tubes
are sterile (autoclaved), and you habitually use RNase-free technique (gloved hands,
autoclaved or oven-baked reagents & glass+plasticware).
2) The best way to store RNA is as an EtOH precipitate -- i.e., in 70% EtOH -- at -20
or -70 deg. To use, mix to resuspend, then remove the volume needed and spin down
DO remember that using gobs of RNases elsewhere in your lab -- such as for plasmid
preps -- may create contamination. If you want a REALLY RNase-free lab, handle
RNases as you would radioisotopes: work on a disposable benchliner & with disposable
gloves; never weigh RNases (or proteases, etc.) on a balance (to prevent traces of
powder from flying loose), avoid any pipeting or vortexing manipulation that could
release aerosols, and generally be aware of what you're doing.
| 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 |
| 2045 Haworth Hall | "The sleep of reason produces |
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