CsCl/EtBr prep: Strange band.

Brian Foley brianf at med.uvm.edu
Wed Nov 24 12:29:04 EST 1993


	The original author did not mention what strain of E. coli he was
using, nor the method of preparing the DNA prior to CsCl gradient
centrifugation, but he noted that he was getting a strange white band that
comigrated with the plasmid DNA.

	The band was only a problem when the culture was grown in terrific
broth, not in LB.

	Page 1.34 of the second edition of MOLECULAR CLONING: A Laboratory 
Manual (Sambrook, Fritsch and Maniatis) states that E. coli strain HB101
and its derivatives will produce copious amounts of a carbohydrate that
will band with plasmid DNA in CsCl gradients.  They say that it is more of
a problem with rapid boil techniques than with alkaline lysis.
	Thus I suspect that your "stange band" is carbohydrate.  It may
not interfere with some things, but it will interfere with sequencing.

	The rule of thumb is:  If it works one way do it that way.  If the
protocol says to use LB media, then use LB media unless you have the time
to test out the new media.  Terrific broth is good for some protocols but
not for others.  Likewise no one strain of E. coli is good for everything.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



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