RNA extraction yeast

Douglas Rhoads DRHOADS at MERCURY.UARK.EDU
Fri Mar 29 08:53:10 EST 1996


> To:            yeast at net.bio.net
> From:          k.b.andersson at biokjemi.uio.no (Kristin B. Andersson)
> Subject:       RNA extraction yeast
> Date:          Fri, 29 Mar 1996 10:50:37 +0100

> Hi!!
> 
> Does anyone have a reliable protocol for RNA extraction from S. cervisiae,
> (small scale preps) for looking at plasmid-derived transcripts??
> 
> Has anyone tried out Triazol??
> 
> Thanks for any info!!!
> 
> -- 
> Kristin B. Andersson, PhD                      k.b.andersson at biokjemi.uio.no
> University of Oslo
> Institute for Biochemistry                Phone:  +47-2285-6699
> PB 1041 Blindern                          Fax:    +47-2285-4443
> N-0316 Oslo, Norway
> 
> 

Here is a protocol that we have found to work on a variety of yeast 
species and that can work on recalcitrant cells.  The prep produces 
fairly clean RNA with little DNA contamination.  For cDNA preps we 
recommend that you run the purified RNA over a size exclusion column 
to remove small molecules below 100 bp.

Isolation of RNA by phenol freeze-thaw
modified from: Schmitt, Brown and Trumpower (1990) NAR 18(10):3091-2
1.   Generate log phase yeast cells (about 10 ml of culture)
2.   Collect cells by centrifugation 5' 2000 x g.  Phillip Stafford has found that for hard to
     crack cells (mycelial Candida) you should not exceed 50  l of packed cells per 400  l
     of AE in step 3.
3.   Resuspend in 400  l of AE buffer (50 mM NaOAc pH 5.3, 10 mM EDTA)
4.   Transfer to microfuge tube and add 40  l 10% SDS and vortex
5.   Add 500  l phenol (pre-equilibrated with AE buffer) and vortex
6.   Incubate at 65oC for 5-10 minutes then vortex
7.   Place at -80oC for 4 hours.
8.   Centrifuge at 10000 rpm for 10'
9.   Collect upper aqueous phase and extract aqueous with equal volume of CHCl3:IAA.  Spin
     and collect aqueous.
10.  If the next step is to make first strand cDNA then add 3 M KOAc to bring to 0.3 M. 
     If RNA is to be purified on oligo-dT or used for northerns then use 3 M NaOAc.
11.  Add 2.5 volumes of ethanol, mix and incubate at -20oC for 1-4 hours.
12.  Collect RNA by centrifugation at 10,000 rpm for 15-20' at 4oC.  Rinse pellet with cold
     70% ethanol.  Dry and redissolve in 20-50  l TE.  Quantitate by spectrophotometry.
yields range from 60 to 300  g RNA per 10 ml culture.  Procedure has been scaled up to 40 ml
(with proportional increases in treatment volumes) for C. albicans.  
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||                  Douglas Rhoads                        ||                             ||
||drhoads at mercury.uark.edu || Dept. of Biological Sciences||
||drhoads at comp.uark.edu    || 601 Science Engineering     ||
||501-575-3251             || University of Arkansas      ||
||FAX 501-575-4010         || Fayetteville, AR 72701      ||
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