Phenol Extraction

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Wed May 7 12:37:22 EST 1997


At 07:46 5/7/97 -0700, Dom Spinella wrote:
>Patrick writes:
>
>> Why do we use acid phenol for RNA extraction and basic phenol for DNA 
>> extraction??
>> Thanks for the answer.
>
>At acidic pH, DNA is selectively retained in the organic phase and the
>interphase, leaving RNA in the aqueous phase and affording a measure of
>purification from DNA.  Also, the acidic pH reduces the activity of many
>nucleases.  Hence, low pH for RNA extractions.  At basic pH, both DNA
>and RNA partition into the aqueous phase. However, for DNA extractions,
>you need to avoid acid-induced depurination; thus, high pH for DNA
>extraction. Hope that answers your question.  -- D.G. Spinella

Dom is correct here.  The basis of the low pH phenol is explained in the
protocol of:
 
Chomczynski,P; Sacchi,N (1987): Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,
156-159.

Here, only water saturated phenol is used here.  This phenol is not pH'ed,
so the Na-acetate which is pH 4.0 dictates the pH of the RNA extraction.
Tris buffered phenol can be used, BUT, I have personally found the RNA
yeilds less and with the higher pH there is a propensity to co-extract a
small amount of genomic DNA from the cells (this has a nasty habit of
complicating Northerns...).  So we have two bottles, one that is RNA only
and the other is >ph7.8 and is for DNA only.

For the "regular" Chirgwin protocol, buy the time you do the phenol
extraction, it doens't manke any difference if one wishes to do the extra
step.

Hope this helps,
David


















=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
" Sometimes you're the windsheild, sometimes you're the bug."
=============================




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