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
>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.
>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.
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