Plasmid DNA Extraction

Lixin Zhou U09723 at uicvm.uic.edu
Tue Dec 29 23:11:12 EST 1992


I got troubles in extracting plasmid DNA from Streptococcus
pneumoniae (Gram-positive).  The plasmid is about 15 kb.
Pneumococcal chromosomal DNA and plasmid DNA are AT rich (about
62%).  The method I used is a modified alkali lysis protocol.
Solution II contains 1% SDS and 0.175 N NaOH instead of 0.2 N NaOH.
Solution III contains 50 mM Tris.Cl-pH8.0 in addition to 0.1 M
NaAc-pH4.8.

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).  Even after I treated DNA from CsCl
prep with Solutions I, II and III for 2-3 times, I still could not
get rid of smear DNA.  Those results seem to be very reproducible.
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
as used in extracting the 15-kb recombinant plasmid.  The recombinant
plasmid contains 10+-kb chromosomal DNA insert from a derivative strain
of the host.

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
Department of Biological Sciences (M/C 066)
University of Illinois at Chicago



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