Help need for Single-step RNA Ioslation by Acid Guanidinium

Randall J. Cohrs COHRS-R at Oberon.HSC.Colorado.edu
Mon Oct 17 16:28:44 EST 1994


> Hi, everybody:
> I deseperately need help for "Single-Step Method of RNA Isolation by Acid
> Guanidinium Thiocyanate-Phenol-chloroform Extraction". I got this protocol from
> the "RED BOOK" and the original paper-Anal.Biochem 162, P156-159,by Chomczynski
> and Sacchi-as the method described proteins and DNA fragment will goes to
> phenol chlorform phase while RNA stay in aqueouse phase after 10000g for 20min
> at 4C. But in my hand the phase nver separate! I check the condition of all
> buffers couple times and still not able to make it work. If any body know
> the possible reasons or met similar problem, Please help!
> 
> Thanks in advance!
> 
> PS: I notice that one component is different in lysis buffer recipy. In the
> original article it's buffered by 20mM sodium citrate PH7, while in "The RED 
> BOOK" it's 25 mM sodium acetate, PH5.5.
> 
 - - - - - 

I had the exact same problem and traced it down to not adding enough 
chloroform:isoamyl alcohol to the extract.

Nothing lost - - just add more chloroform:ios-oh , vortex  and the phases will
seperate.  If you have anyother problem , re-post and we'll take it 
from there


rjc in denver 



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