Plasmid DNA Extraction

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


In article <92364.221112U09723 at uicvm.uic.edu>, Lixin Zhou <U09723 at uicvm.uic.edu> 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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