bertheau@jouy.inra.fr (Yves Bertheau) (04/26/91)
We have some troubles during the course of a genomic substraction with ligation Does somebody try ligation of oligonucleotides adaptators (one phosphorylated at PCR using one strand of the adaptataors as a primer ? We were trying to use Ausubel's protocol... Any help would be appreciated. Thanks in advance. Yves Bertheau, Pathologie vegetale, INRA, PAris France.
darrasse@inapg.inra.fr (Armelle Darrasse) (04/30/91)
> > Forwarded message: > > >From bwoodman@magnus.acs.ohio-state.edu Mon Apr 29 15:58:59 1991 > Date: Mon, 29 Apr 91 09:49:21 -0400 > From: Robert H Woodman <bwoodman@magnus.acs.ohio-state.edu> > Message-Id: <9104291349.AA27768@bottom.magnus.acs.ohio-state.edu> > To: bertheau@jouy.inra.fr > Subject: Re: Genomic substraction > Newsgroups: bionet.molbio.methds-reagnts > In-Reply-To: <9104270238.AA14321@genbank.bio.net> > Organization: The Ohio State University > > In article <9104270238.AA14321@genbank.bio.net> you write: > >We have some troubles during the course of a genomic substraction with ligati > >Does somebody try ligation of oligonucleotides adaptators (one phosphorylated > >PCR using one strand of the adaptataors as a primer ? > >We were trying to use Ausubel's protocol... > >Any help would be appreciated. > >Thanks in advance. > > >Yves Bertheau, Pathologie vegetale, INRA, PAris France. > > Yves, > > Hi! Could you be a bit more explicit in the kind of problems you are > having and/or think you might be having? Also, could you reference Ausubel's > protocol. I haven't yet tried a genomic subtraction, but I'm supposed to be > doing such sometime in the near future. > > Thanks, > > Bob Woodman > > > -- > ********************************************************************* > *Robert H. "Bob" Woodman, PhD * "A job not worth doing well is not * > *INTERNET: woodman.1@osu.edu * worth doing."--Salvador Luria * > ********************************************************************* > > > -- > > Yves Bertheau > INRA INA P-G > Pathologie Vegetale > 16 rue Claude Bernard > 75231 Paris cedex 05 > FRANCE > Phone: +33 (1) 43.31.93.97 Fax : +33 (1) 43.31.83.82 > Telex: 250985 Internet bertheau@inapv.inra.fr > We are currently trying to do a genomic substraction according to the protocole of Ausubel (Straus D., Ausubel F. M., Genomic substraction for cloning DNA corresponding to deletion mutations, Proc. Natl. Acad. Sci. USA, Vol. 87, March 1990). To summerise, one DNA (containing sequences that are absent in the other one), called ``the target DNA'', is cut by a restriction enzyme and hybridized several times with the second DNA (sheared by sonication and biotinylated). Biotinylated DNA and complementary sequences (from ``the target DNA'') are removed with streptavidin beads. Only the non- homologous sequences are conserved. They are present in small quantities. Then we have to ligate these DNA fragments with oligonucleotide adaptators (compatible with the restriction site). These adaptators are made with 2 complementary oligonucleotides : one has to be phosphorilated at the 5' end. After this, the oligonucleotides are mixed and hybridized together. The DNA fragments are capped with the adaptators (by ligation). This step allows an amplification by PCR, using one strand of the adaptator as primer. We have some problems to ligate successfuly adaptators to the DNA fragments and obtain after PCR something else different than a smear (with a molecular size much larger than expected...). Has anybody any experience with this protocole and would care to help? Thanks. -- Armelle Darrasse INRA INA P-G Pathologie Vegetale 16 rue Claude Bernard 75231 Paris cedex 05 FRANCE Phone: +33 (1) 43.31.93.97 Fax : +33 (1) 43.31.83.82 Telex: 250985 Internet: darrasse@inapv.inra.fr