Problems with Pseudomonas putida minipreps

[Jason Brinck]

 Hi all,

Does anyone have any good ideas to help me get round the following problem:

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 have tried different miniprep methods (alkaline lysis, boiling) as well as
kits (Bio101 RPM kit) but seen little improvement. I know that this strain is 
not very "user-friendly". It has a low viability on plates (up to a week) so I 
was wondering if part of the problem is how I store the samples until I 
miniprep them all at the end of the experiment. Might storing them at -70 C 
improve things. Other than that all I can think of is storing the sample as a 
glycerol but I think that would then cause problems when I come to miniprep 
the samples.

Even so, I'm still not happy with the miniprep method as I still get uncut 
bands when I miniprep fresh culture and linearise it.

So, any suggestions would be greatly appreciated as time is running out for me!

Jason Brinck,
School of Biochemistry
Birmingham University, UK