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Help: mRNA purification

Doug Rhoads DRHOADS at MERCURY.UARK.EDU
Mon Jun 20 07:49:20 EST 1994


>------- Forwarded Message Follows -------
>From:           skaja at aries.scs.uiuc.edu (Anne Skaja Robinson)
>Date sent:      20 Jun 94 01:48:42 GMT
>
>I haven't seen a FAQ on this, so if there is one, please let 
>me know.
>I am interested in information on mRNA purification from S. 
>cerevisiae.  I have seen several protocols using hot phenol
>and mechanical lysis, but I was hoping to find a better 
>technique.  Particularly if anyone has used Tri-Reagent, I
>am interested in their success.
>Thanks a lot!
>Anne Robinson

I would also be interested in discussing this thread.  We have 
experimented with using freezing in acidic phenol, as well as hot benzyl 
chloride.  Both work pretty good on actively dividing budding cultures.  
But, we don't work with Saccharomyces and many of our cultures are in a 
mycelial state.  The mycelial cultures usually have a low yield and most 
preparations are heavily contaminated with very low molecular weight, UV 
absorbent, ethidium stainable material.  The material can be removed by 
size fractionation on spin columns (not cost effective for hundreds of 
samples).  Our experience suggests a window of cell mass for the acid-
phenol freeze thaw.  The benzyl chloride lysis (NAR 21:5279-80) was 
modified from a DNA isolation protocol and does have some DNA 
contamination.

Doug Rhoads                  || Dept. of Biological Sciences
drhoads at mercury.uark.edu     || 601 Science Engineering
drhoads at uafsysb.uark.edu     || University of Arkansas
501-575-3251                 || Fayetteville, AR 72701



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