Extracting DNA from fixed tissue?

Peter Thorén peter.thoren at genetik.uu.se
Thu May 26 07:10:53 EST 1994


In article <2s0t2a$449 at molbio.usc.edu> zarow at molbio.usc.edu (Chris Zarow) writes:
>From: zarow at molbio.usc.edu (Chris Zarow)
>Subject: Extracting DNA from fixed tissue?
>Date: 25 May 1994 18:12:42 -0700

>Greetings to all.

>     I have been trying to extract genomic DNA from formalin
>fixed human cerebellum. Since I am having no luck, I welcome your
>insights and suggestions.
>     I started out using a standard DNA extraction protocol
>(Short Protocols in Molecular Biology) where the tissue (about 1
>gram) is homogenized in 10 volumes of 100mM NaCl/10mM Tris pH 8.0/25
>mM EDTA/ 0.5% SDS/ 0.1 mg/ml proteinase K.  (I am using a Tissue
>Tearor mechanical homogenizer.)  The homogenate is incubated
>overnight at 50 degrees C.  Following two phenol/chloroform
>extractions, DNA from the aqueous phase is precipitated with ethanol.
>     Although I have used this method with good results for
>unfixed human cerebellum, I have obtained zero DNA from fixed
>tissue.
>     After the standard method failed, I consulted some protocols
>which describe methods to run PCR on DNA from paraffin-embedded,
>formalin fixed tissue. I proceeded from the premise that formalin
>fixed tissue has not been processed like paraffin-embeded tissue
>has, and therefore the DNA should be easier to extract. On the
>assumption that the proteinase K might be being inhibited or
>inactivated by the formalin, I tried washing the tissue overnight
>in 1000 volumes of running water to remove the formalin.  Then I
>added 0.1 mg/ml proteinase K to the homogenate every 2 hours, to
>a final concentration of 0.5 mg/ml at 50 degrees C. Still no DNA.  I
>also tried to extract DNA using a boiling method from one of the PCR
>papers.  Still no DNA.
>  
>     One observation which might give someone some insight is
>that the white interface following the phenol/chloroform
>extraction is much thicker with the formalin tissue than with the
>fresh tissue.  I can't help but think that the DNA is somehow
>still complexed with protein and is trapped in the interface, but
>I'm not sure how to get it out. 
>  
>     I would appreciate receiving any comments/ suggestions/
>insights/ solutions to this problem.  Thank you.

>Chris Zarow, Ph.D
>University of Southern California
>-- 
>Chris Zarow, Ph.D
>University of Southern California
>Rancho Los Amigos Medical Center
>zarow at molbio.usc.edu

Have you tried the Chelex-100 method? If you want the reference I have it 
somewhere in my room if you want it. I have used this method for isolating DNA 
from bumblebees and it´s really quick and cheap.

Peter Thorén
Peter.Thoren at genetik.uu.se






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