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.
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
Institute of Applied Microbiology
holubar at mail.boku.ac.at