rothstei at calvin.jci.tju.edu
Mon Dec 13 08:52:26 EST 1993
In article <2D0C3BC0.554 at news.service.uci.edu>, rwcitek at orion.oac.uci.edu
(Robert Citek) wrote:
> I need to work with RNA and have looked in several sources to
> minimize or eliminate RNAse contamination. DEPC was recommended
> in 0.2%, 0.1%, 0.5%, and 0.001% concentrations to treat my water
> for preparing buffers. I've never worked with RNA before. Does
> it really matter what concentration I use? That is, is 0.2% going
> to be so high that it will interfere with later RNA use or is 0.001%
> going to be so low as to be ineffective?
> Thanks in advance for any and all advice.
> Robert Citek
> University of California
> 134 Social Ecology
> Irvine, CA 92717-5150
> rwcitek at uci.edu
We have worked with RNA for many years and it is not necessary to
DEPC-treat at all. We have a milli-Q water filtration system and find that
all water from this unit is RNAse free (Over a 4 year period we never once
saw RNAse activity in our water stocks). What we do is test all reagents
before use by combining them with a high mol. weight RNA (such as BRL's 7.5
Kb RNA) incubate at 37 deg. for 15-20min and and resolve on a northern gel
(short run just to get the RNA in the gel maybe 100 volts for 30-45 min).
The results are viewed by placing the gel on plastic wrap and on top of an
intesifying screen. A hand held UV light is then held abve the gel and RNA
bands can be seen as "shadows" If any degradation has occured the lane
will appear empty or smeared. This is the best way to test for RNAse
activity while avoiding the carry over of DEPC into reactions.
In 6 years I have not had to DEPC-treat any reagents and we routinely
collect small amounts of precious RNA from mouse embryos in this manner.
(Actually the amount of DEPC I have heard others use was 0.001% incase you
still want to do it that way)
Jay L. Rothstein, Ph.D.
Jefferson Cancer Institute
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