We've been surprising succesfull, even with 32- to 35-mers,
in screening with unpurified DNA straight from IDT. We
generally try purifying the oligos that give us hits, but it
often doesn't improve the crystals.
Sounds shockingly sloppy, but saves lots of time and $$!
Phoebe Rice
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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 isn4t.
>
> with regards
> Rajesh......
>
>
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