How to clean DNA which may have glycosaminoglycans

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Wed Aug 20 13:52:09 EST 1997


Dear Warren --
	Thanks.  I DID try CTAB, but I used a completely different 
concept than what I found in Current Protocols.  I squirted my DNA 
directly a CTAB solution with low salt, and the DNA fell out of 
solution.  I then took my DNA back up in 2 M NaCl, and finally diluted 
down the salt, ethanol precip, and the resulting DNA still was 
uncuttable.
	Current Protocols (under bacteria 2.4.2 supplement 24) gives a 
protocol for "removal of polysaccharides from existing genomic DNA 
preps" in which the DNA is brought up to ~700 mM NaCl, has added CTAB 
added to 1%, and then the DNA is extracted a few times by chloroform, 
followed by isopropanol precipitation.  I have done this today, will 
digest, and let you know how it comes out.
	Thanks, 
	Harry

Warren Gallin wrote:
> 
> In Article <33FA1F8E.655D at Bristol.ac.Uk>, Harry Witchel
> <Harry.Witchel at Bristol.ac.Uk> wrote:
> >Good day Molecular Charmers!
> >        I have a problem with my DNA.  Boy do I have a problem!  My DNA
> >comes from a marine invertebrate which is hypocellular and filled with
> >gelatinous mesoglea which I'm told is made up of glycosaminoglycans or
> >hyaluronic acid.  This thing is mostly connective tissue.
> >        I have made the DNA, the animals are long gone, I have a limited
> >quantity.  I have phenoled, chloroformed, ethanoled and isopropanoled
> >till blue in the face, but still, the stuff does not digest well with
> >restriction endonucleases.  Even after running down the purified DNA
> >through Qiagen's genomic column, this DNA does not want to cut to
> >completion.
> 
> The two methods that have worked for me are 1) CTAB purification and 2)
> Chelex purification.  I believe that you will find both of these in the
> Current Protocols in Molecualr Biology book, probably in other manuals.  If
> not, drop me a line and I'll dig out my files and find a procedure for you.
> 
> Warren Gallin
> Department of Biological Sciences
> University of Alberta
> Edmonton,  Alberta     T6G 2E9
> Canada
> wgallin at gpu.srv.ualberta.ca



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