How to separate plasmid DNA from Chromosome DNA?

Song Tan tan at mol.biol.ethz.ch
Thu Dec 1 04:10:35 EST 1994


In article <Stephen_Lasky-2911941447280001 at poncho-slip11.cis.brown.edu>,
Stephen_Lasky at brown.edu (Stephen R. Lasky, Ph.D.) wrote:

> In article <29NOV199411342814 at rosie.uh.edu>, chee1tq at rosie.uh.edu (Xu,
> Liang) wrote:
> 
> > Is there any good way to separate chromosome DNA from plasmid DNA?
> > Ony way I kown, is to use ultracentrifuge, since the molecular weights
between
> > plasmid DNA and chromosome DNA are different. But for the large scale
> > plasmid DNA production up to couple hundred milogram, it is not a very
> > good and efficent to use ultracentrifuge. Does anybody have good thought?
> 
> The standard alkaline lysis technique with KOAc precipitation does just
> this if you are carefull to not shear the chromosomal DNA. [snip]

As Stephan Lasky has indicated, alkaline lysis should eliminate
chromosomal DNA.  However, if there is any chromosomal DNA leftover that
you really want to get rid off, you can use a Sephacryl S1000 column to
fractionate the chromosomal DNA, plasmid DNA and any RNA you might have. 
And this can be scaled up to hundreds of milligrams of DNA.  A reference
from our lab for such a large scale plasmid prep can be found in:

Richmond et al., Crystals of a nucleosome core particle containing defined
sequence DNA, JMB,
199, 161-170, 1988.

Another interesting reference for large scale plasmid preps is:

Hillen et al., Preparation of milligram amounts of 21deoxyribonucleic acid
restriction fragments, Biochemistry, 20, 3748-3756, 1981.

Good luck!

-- 
Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email:  tan at mol.biol.ethz.ch



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