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

BAI_JN at JHUVMS.HCF.JHU.EDU BAI_JN at JHUVMS.HCF.JHU.EDU
Mon Oct 17 13:17:09 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.



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