Plasmid DNA Extraction

Peter M. Muriana muriana at
Wed Dec 30 11:30:33 EST 1992

In article <92364.221112U09723 at>, Lixin Zhou <U09723 at> writes:
>I got troubles in extracting plasmid DNA from Streptococcus
>pneumoniae (Gram-positive)......
>The troubles were: in both minipreparation (1-10 ml culture) and
>big-CsCl-preparation (3 Liters), I always could not separate
>plasmid DNA from 'chromosomal DNA' (or whatever else?).  In CsCl
>centrifugation tubes, I always got only one band which appeared not
>sharp (50Ti rotor, 48K rpm for 50-60 hrs, or even longer).  When I
>run DNA from that band on agarose gel, I saw both plasmid and
>'chromosomal DNA' (It appears to be chromosome or from chromosome.
>I will called it smear DNA)..........

>I repeated CsCl-preparation three times and miniprep several times.
>Every time, I could not separate plasmid DNA from cloudy DNA.
>Reagents including 2 N NaOH stock seem to work fine.  I successfully
>extracted 4.4-kb vector DNA (father of the 'un-extractable' plasmid)
>from a different strain of the same species by using the same reagents
    The "different strain" may not have the peculiarity of the strain in 
    question.  Don't Streptococcus pneumoniae make polysaccharide capsules?
    Perhaps this may be part of the problem? 	

>Could anybody see and tell me why I failed the separation?  What might
>the smear DNA be?  Thanks in advance.

>L Zhou
>Laboratory for Molecular Biology

	This may seem like a silly question to your question, but you are
	running Ethidium Bromide-CsCl gradients, not CsCl gradients, right?

	I experienced a problem with various Gram-positive genera in my 
	past where there was so much contaminating protein, etc., that
	went into the EtBr-CsCl gradient, that they reduced the level of
	EtBr available to intercalate into the pDNA/chrDNA, thereby not
	allowing effective separation of pDNA from chrDNA.

Peter Muriana

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