Invitrogen's pZERO & E.coli with ccdA
malexe at biost1.thi.tmc.edu
Tue Aug 13 14:31:54 EST 1996
In article <malexe-1308961220250001 at 188.8.131.52>,
malexe at biost1.thi.tmc.edu (Mikhail Alexeyev) wrote:
> In article <4uq75r$hrh at frazier.backbone.uoknor.edu>,
> bsellman at microbiology.bmsb.uokhsc.edu (Bret Sellman) wrote:
> > I have been using the pZERO vector from invitrogen with great success.
> > The only problem is that the vector is not cheap to buy and I cannot
> > propagate the plasmid without first cutting it and ligating in DNA.
> > Does anyone know how to propagate this vector? Are there any E. coli
> > strains available which overexpress ccdA. Thanks.
> In the original article authors used some gyrA (gyrA 401?) mutant to
> propagate plasmids (gyrA96 mutation common to many laboratory strains will
> not do the job). The article has been published in Gene, so strain should
> be available from authors. I am not aware of any ccdA -overexpressing
> strains, but it should be possible to construct one (the gene is encoded
> by F plasmid -just PCR it out, place under the strong promoter in the
> transposon and shoot it into the chromosome). On the other hand, if lac
> promoter is repressed well on the plasmid, one should be able to propagate
> plasmid in a strain bearing F'.
> Does Invitrogen still require to sign a peper saying that you are not
> going to attempt to propagate vector without an insert?
Sorry for self-followup. Just received an August issue of BioTechniques
with an article by Philippe Bernard on the subject. Look at the page 320.
More information about the Methods