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
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!
School of Biochemistry
Birmingham University, UK