DNA extraction from P. fluorescens

Darren.Martin Martin at gene.unp.ac.za
Mon Jun 27 09:20:48 EST 1994


In article <2u69c0$2ct at osiris.wu-wien.ac.at> seb at i102pc1.vu-wien.ac.at (Sebastian W. Bunka) writes:
>From: seb at i102pc1.vu-wien.ac.at (Sebastian W. Bunka)
>Subject: Re: DNA extraction from P. fluorescens
>Date: 21 Jun 1994 08:45:52 GMT

>Ron Caspi (rcaspi at ucsd.edu) wrote:
>: Hello Netters

>: I am working with a strain of Pseudomonas fluorescens, and when I try to 
>: extract chromosomal DNA, I am faced with large amounts of polysaccharides, 
>: that make life much more complicated. Does anybody have suggestions, either a 
>: different growth medium or a protocol that will solve the problem?

>: Ron

Hi Ron

I had exactly the same problem with the strain of Ps. fluorescens that I was 
trying to extract DNA from.  I had to resort to doing mini-preps of 
chromosomal DNA in eppendorfs (I did get quite a lot though - +- 80 ug 
from six preps each using 5ml of an OD600=0.6 culture).  When extracting 
with chloroform/phenol I removed the organic phase/proteinacouse precipitate 
from the tubes first befor transferring the aqueouse phase to a fresh 
tube using a one ml micropippette with the end of the tip cut off to give 
it a wider bore. The "gooieness" of the aqueouse phase remained until 
phenol/chloroform was extracted with ether (2 treatments fro every 
phenol-chloroform treatment).  The DNA I obtained was really clean and 
could be restricted and ligated.    

Reply directly to me for any extra details.

Darren



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