mRNA isolation in E. coli

gordon allison goa at aber.ac.uk
Tue Feb 6 11:25:47 EST 1996


In article <31159EF3.701F at leland.stanford.edu>, rslany at leland.stanford.edu 
says...
>
>Rafael Maldonado wrote:
>> 
>> Hi netters!
>> 
>> I need a good, reliable protocol for RNA isolation in E. coli. Since I
>> am interested in a time course of amount of mRNA in a culture, it would
>> be ideal a method which used low amount of cells (1 ml) to get enough
>> mRNA for a primer extension reaction (i. e. 10-40 ug). Any suggestions
>> welcome.
>> 
>> TIA
>> 
>> Rafa
>> 
>> ___________________________________________________________________
>>                                              |
>> Rafael Maldonado                             | 'No te creas todo
>> room 6160 Eccles Institute of Human Genetics |  lo que leas en
>> Department of Human Genetics                 |  internet.'
>> University of Utah                           |
>> Salt Lake City, Utah 84112. USA.             |  "Don't believe
>> Rafael.Maldonado at genetics.utah.edu           |  everything you read
>> Rafael at howard.genetics.utah.edu              |  in internet."
>> Tel: 801-581-4429                            |
>> Fax: 801-585-3910                            |
>> 
>
>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


I used to prepare cyanobacterial RNA by microwaving cell suspensions in 
SDS, followed by hot acid phenol and chloroform. However, I have mellowed 
as the years have passed and now opt for the more moderate approach of 
Qiagen RNeasy, it's like "wash and go", all you need in one kit and it 
only takes 10mins.

good luck, Gordon




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