Miniprep method - Why the mild conditions?

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Fri May 16 13:46:50 EST 1997


Hi buggers!

Why is there a need for such a mild procedure?
It doesn't seem that it would neither save 
time nor money. There are procedures to avoid 
costly spin columns, but at least the one I'm 
using does it with alkaline lysis.

Cheers, 

Wolfgang 

In article <jmejia-1305971753430001 at larin1.imm.ox.ac.uk>,
jmejia at worf.molbiol.ox.ac.uk (Jose E. Mejia) wrote:

> > > Hello,
> > > 
> > > I am looking for protocols or kits fo plasmid minipreps that do
> > > not involve denaturation of DNA, i.e. neither alkaline nor
> > > boiling lysis. If anyone knows about any such methods, please
> > > drop me a line.
> > > 

Zinder & Boecke describe such a method in Gene 19 (1982) 1-10

It is rather obscure, in the methods section (if I remember correctly)
and don't be confued by the fact that they are purifying dsM13 DNA
(which is by all practical means just a low copy number plasmid).

The procedure is very mild.  The bugs are lyzed with Lysozyme on ice.
There is a heating step at 65*C to denature protein but that should
not be a problem,  PEG precipitation,  RNase digestion,
Pheno/chloroform extraction etc.

This takes some time but gives good yields of pure and almost 100%
supercoiled plasmid if done correctly.

Zophonias

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Zophonias O. Jonsson Institut fur Veterinarbiochemie              
Tel: (41-1)-257-54-75 Universitat Zurich-Irchel                    
Fax: (41-1)-362-05-01 Winterthurerstrasse 190 CH-8057 Zurich
Switzerland
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University of Tuebingen
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