Dr. Oliver,
I have also experienced poor yields when isolating _P. putida_ plasmid DNA,
which tend to be large in size and low in copy number. Large scale CsCl
gradient isolation works nicely, but is time/labor intensive. The general
protocol below has worked well for me:
http://www.nwfsc.noaa.gov/protocols/lc_plasmids_qia.html
I have had success amplifying the plasmid copy number (per Maniatus) by
adding 25 ug/mL chloramphenicol (or spectinomycin) when the O.D. = 0.6. Ray
Scheetz (rfs123 at psu.edu) has performed large scale psuedomonad plasmid
isolations (for soil persistence work) and would be a helpful source of
additional information.
P.S. I admire your work with VNC organisms. Are you looking for another
skilled, motivated Ph.D. student at UNC?
John Hegarty M.Sc.
jph10 at psu.edu
Water-borne _Helicobacter pylori_
http://www.personal.psu.edu/jph10
Environmental Microbiology Lab
Penn State Harrisburg
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"Dr. James D. Oliver" wrote in message
<3.0.32.19981228135758.0070b100 at email.uncc.edu>...
We are trying to identify a plasmid in <italic>Pseudomonas
fluorescens</italic>. We have used a variety of "typical" mini-preps,
including some commerical ones, with little to no luck. We realize most
prep methods are designed for E. coli, so would appreciate hearing from
anyone who can offer assistance (a different method?) for isolating
plasmids from pseudomonads. Thanks much!
