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We recently solved the structure of a protein bound to a 21bp dsDNA ligand using DNA bought from idt (idtdna.com), standard desalting preparation. After mixing the dsDNA and protein, we isolated the complex with gel filtration. Also, removing the His-tag from the protein was important. We've also had success crystallizing a Fab complexed with shorter ssDNA oligos (3-10 bases), with the DNA again bought from idt (standard desalt). In the Fab/ssDNA cases, we used the ssDNA in 3-5 fold excess and didn't do gel filtration. Good Luck, Jack -- John J. Tanner Professor of Chemistry and Biochemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: [EMAIL PROTECTED] http://www.chem.missouri.edu/TannerGroup/tanner.html > From: ponnusamy rajesh <[EMAIL PROTECTED]> > Date: Fri, 22 Sep 2006 11:32:10 +0200 > To: <[EMAIL PROTECTED]> > Cc: <[EMAIL PROTECTED]> > Subject: [ccp4bb]: Purity of DNA for protein-DNA complex > > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Dear folks, > > Again, a experimental question. I am working on protein DNA > complex. the protein which i am working binds to the DNA unspecifically > and i do have the native form of the strcture. Since, then i have been > trying to do DNA-protein complex. > Since, the protein binds unspecifically to DNA. The trial of > experiments goes exponentially higher, with that the cost of the > experiment also goes higher :-( . > There was lots of discussion earlier about the length of the DNA, sticky > ends, overhangs...etc. > > But, my question comes from the area about purity. > How pure the DNA should be for the crystallization trials? > > I used to buy DNA without much purification, This is as normal as > buying primers for cloning. So, the purity is not much high. > for eg. there are purification like > Hydrogel Purity Electrophoresis, High Performance Liquid Chromatography, > which i am not using. > > So, how good the purity should be for intial trials? > might be in later case i could go for high purity DNA during the > optimization isn´t. > > with regards > Rajesh...... > >
