RNA extraction from e. coli for northern

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Feb 8 07:49:03 EST 2000


In article <389FF219.4DB73AD5 at gmx.net>, Frederik Börnke
<ricky_boernke at gmx.net> writes
>does anybody have a quick n' easy protocol for extraction of
>RNA from E. coli? I'd like to do some northern ananlysis.
>
>TIA
>Ricky
>
>PS I already looked up the archive, but nothing found.


>From the archive many moons ago:


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Dear Rafael,
a former collegue of mine has worked out a very nice protocol for
isolation of total RNA from E.coli. Since there is no polyA tail on this
RNAs there is no possibility to select for mRNAs. The problem is that
the typical half-life of bacterial mRNA is about 2 minutes!! So it is
essential to inactivate all cellular processes b e f o r e you harvest
your bacterial cultures. If you spin your cultures down, the RNA
composition will change substantially during 
the 10 min spinning time.

Isolation of total RNA from E.coli:

Grow 8ml bacterial culture to mid-log phase (O.D. app. 0.7)
Pour whole culture quickly into 16 ml of ice-cold 80%Ethanol/1%DEPC
Centrifuge 10min, 6000 rpm
Resuspend in 1ml of (Guanidiniumisothiocyanate 20g, water 21,3ml, 1M 
Tris-HCl pH 7.5 2.2ml, 0.5M EDTA 850μl, 20% Na-Lauroylsarcosinate 4.2
ml, immediately before use add 500μl b-mercaptoethanol)
Heat 3min to 90C, mix gently
Add 0.1g CsCl, mix gently, heat again to 90C (get a highly viscous
solution)
Layer this crude preparation on top of 1ml of a 5.7M CsCl cushion (in
water) spin in swinging bucket rotor app. 12h at 32000 rpm and 18C.
(Sorry, I've no idea how many xg this is, but should be pretty much the
same in any usual normal sized rotor)
Next day: suck off supernatant (contains DNA and protein at interface),
RNA will be a transparent pellet at the bottom of the tube.
Dissolve RNA in TE or water, extract with phenol till you don't see any 
protein at the interphase any more and precipitate.
Expect several hundred micrograms of RNA which will be >85% ribosomal
and tRNA.


This is kind of lenghty and seems circumstantial but it works.
Good luck.

Robert

P.S. Any credit for this method should go to Michael Boesl


-- 
Robert Slany, Dept. of Pathology, Stanford School of Medicine, Stanford 
University
300 Pasteur Drive, Stanford, CA, 94305, USA, Phone:001-415-725-5696, 
Fax: 001-415-725-6902,  
rslany at leland.stanford.edu

------------------------------------------------------------------------

Alternatively go back through the last 3 or 4 years of Biotechniques.

Duncan

-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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