Pseudomonas transformation

Stephan Heeb Stephan.Heeb at Lbm.unil.ch
Mon Feb 5 08:48:57 EST 1996


In article <4f0gs0$6no at netnews.upenn.edu>, pnealen at mail2.sas.upenn.edu
(Paul M. Nealen) wrote:

> I am seeking information on transformation of Pseudomonas - I know that 
> 37 degrees and Ca++ are used, as is 37 degrees and Mg++.  Have any other 
> temperature/ion combinations been used?
> 
> Thanks -

Here you have the protocol we are using for routine tranformation of
Pseudomonas fluorescens strain CHA0.

Transformation of this strain is not possible with electroporation (high
frequencies of mutations were observed) and the temperatures should not
exceed 35°C (the strain won't grow at 37°C). This method is a modification
of the stadard protocol described for E. coli in "Molecular Clonig, A
Laboratory Manual"; Sambrook et al. 1990)

Good luck,

        Stephan Heeb             

        Lab. Biol. Micr.          e-mail: Stephan.Heeb at LBM.unil.ch
        Universite de Lausanne
        1015 Lausanne
        Switzerland

===================

LBM PROTOCOLS
-------------

PREPARATION OF FROZEN STOCKS OF COMPETENT CELLS AND TRANSFORMATION OF
PSEUDOMONAS FLUORESCENS CHA0

Dilute 300 ul of a fresh overnight culture into 30 ml NYB and incubate
with shaking at 35°C (to prevent restriction) until OD600 = 1.8-2.0

Centrifuge 5 min. at 5'000 rpm 4°C in a Corex tube and resuspend cells in
7.5 ml of chilled 0.1 M calcium chloride using a Pasteur pipette.

Leave on ice for 30 min.

Centrifuge 5 min. at 5'000 rpm 4°C and resuspend cells in 1.5 ml of
chilled 0.1 M calcium chloride using a Pasteur pipette.

* Add 50 ul of DMSO, mix gently by swirling, and store the suspension on
ice for 15 minutes.

* Add an additional 50 ul of DMSO to the suspension. Mix gently by
swirling, and then return the suspension to an ice bath.

* Working quickly, dispense 100 µl aliquots of the suspensions into
chilled, sterile Eppendorf tubes. Snap-freeze the competent cells by
immersing the thightly closed tubes in liquid nitrogen or in a dry
ice/ethanol bath. Store the tubes at -70°C until needed.

* When needed, thaw the cells in an ice bath. Store the cells on ice for
10 minutes.

Mix 100-500 ng plasmid DNA with 100 ul of competent cells.

Leave on ice for 30 min.

Heat pulse at 42-43°C for 2 min.

Add prewarmed (35°C) NYB to complete 1 ml and incubate shaken at 35°C for
1 to 3 hrs depending on the plasmid copy number (less time is required for
high copy number plasmids).

Plate on selective media.



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