Miniprep method

Alexander Kraev kraev at bc.biol.ethz.ch
Thu May 15 07:40:48 EST 1997


In article <bbeitzel-1405970727410001 at idl04.salk.edu>,
bbeitzel at jeeves.ucsd.edu (Brett Beitzel) wrote:

> There is a method that was in biotechniques within the last couple of
> years called the "speed prep," which basically involved resuspending the
> bacterial pellet in a LiCl containing buffer and doing a phenol
> extraction.  I've tried it a couple of times without much success, but
> maybe its just me.

No, it was the strain.  However, the boiling lysis with LiCl  (my favourite)
does not actually denature the DNA, it just brings up the temperature
of the Triton solution to about 90 C shortly (the solution in the tube
does not boil)
 which  is enough for the plasmid to go out.  The oldest plasmid isolation
 method (the one before the alkaline revolution) of Clewell and Helinski
 is also a version of the Triton lysis, and  it was also known to be the most
 "gentle" to large DNA, but that one lysed the cell completely and the
 chromosomal clot could only be pelleted at high speeds, not yet achieved
 in compact centrifuges even today.

> 
> 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.
> > 

As mentioned above, boiling lysis in the presence of  2.5 M LiCl does not
actually denature the DNA, it just perforates the cell in the same way as
LiCl-phenol.  As a consequence,  no Freddy plasmid (believed to be
a single stranded circle, often seen after alkaline lysis made at improper cell
concentration)  is observed with it. Finally, some old plasmid screening methods
lysed bacteria in the agarose slot with SDS/proteinase K/EDTA and the
plasmid DNA
could be electrophoresed away from the chromosomal. I think MWG-Biotech here
in Europe sells an apparatus that can be adapted for this purpose. However,
you cannot handle large amounts of DNA in this way, only in the order of
10-100 ug.

I hope this helps,

(Ah, yeah, no affiliation to MWG-Biotech)

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



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