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Problems with Pseudomonas putida minipreps

Peter Holubar holubar at mail.boku.ac.at
Sun Sep 24 11:54:39 EST 1995


J.D.Brinck at bham.ac.uk (Jason Brinck) wrote:


>I routinely grow a strain of Pseudomonas putida, which contains a recombinant 
>plasmid, as a continuous culture. I am trying to analyse plasmid DNA isolated 
>from samples taken at different time points during the experiment.
>
> As well as observing if the size of the plasmid remains unchanged I would 
>also like to see if the intracellular concentration of plasmid varies. In an 
>attempt to do this I have harvested equal amounts of cells at each sample (the 
>equivalent of 1ml of O.D. 2 culture) which I then store at -20 C as a 
>dry pellet. At the end of the experiment plasmid DNA is isolated from all 
>the samples using a standard miniprep protocol.
>
>The problem I have encountered is a complete lack of consistancy / 
>reproducability. The amount of plasmid DNA I recover from minipreps is highly 
>variable, even between duplicate samples. Also, the quality of DNA obtained is 
>often poor such that when I try to linearise the plasmid with HindIII a lot of 
>the samples only partly cut.


Hello,

I don´t know the size of your plasmide, but I once had similar problems 
with a large plasmide in pseudomonads. Large plasmides are often strongly 
associated to the cell wall, so conventional minipreps would not lead to 
sufficient yield of plasmide. Maybe a solution is to digest your cells 
with proteinase K for a longer time than mentioned in the standard 
protokoll.

Peter Holubar
Institute of Applied Microbiology
Vienna, Austria
holubar at mail.boku.ac.at
   




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