Extracting DNA from fixed tissue?

Surasakdi Wongratanacheewin sura_wng at KKU1.KKU.AC.TH
Sat May 28 04:16:01 EST 1994


Dear netters
=09I ever done DNA hybridization using formalin treated sample as a=20
target on nitrocellulose but it fail. I expected that the DNA may be=20
destroyed by formalin till they can't be detect and may also can't be=20
extract! Is there any other comment? I'm also love to know that. Thanks.

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*
S. Wongratanacheewin, Ph.D=09=09=09Khon Kaen University
Department of Microbiology=09=09=09Khon Kaen 40002 Thailand
Faculty of Medicine=09=09=09=09Tel: 66-43-245990
E-mail: sura_wng at kku1.kku.ac.th=09=09=09Fax: 66-43-243064
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On Thu, 26 May 1994, Peter Thorin wrote:

> 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
>=20
> >Greetings to all.
>=20
> >     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.
> > =20
> >     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.=20
> > =20
> >     I would appreciate receiving any comments/ suggestions/
> >insights/ solutions to this problem.  Thank you.
>=20
> >Chris Zarow, Ph.D
> >University of Southern California
> >--=20
> >Chris Zarow, Ph.D
> >University of Southern California
> >Rancho Los Amigos Medical Center
> >zarow at molbio.usc.edu
>=20
> Have you tried the Chelex-100 method? If you want the reference I have it=
=20
> somewhere in my room if you want it. I have used this method for isolatin=
g DNA=20
> from bumblebees and it=B4s really quick and cheap.
>=20
> Peter Thor=E9n
> Peter.Thoren at genetik.uu.se
>=20
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>=20
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