Help need for Single-step RNA Ioslation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction"

David J. Meyer meyerdj at biovx1.biology.ucla.edu
Wed Oct 19 19:28:40 EST 1994


In article <01HIDWGZFJEAHTSRCW at JHUVMS.HCF.JHU.EDU>,
BAI_JN at JHUVMS.HCF.JHU.EDU wrote:

> 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.

Try adding more CHCl3/isoamyl-OH to the homogenate. This may work if the
homogenate is not too viscous.

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David J. Meyer
Department of Biological Chemistry
University of California-Los Angeles
meyerdj at biovx1.biology.ucla.edu



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