Mutation and Sorbitol *******a

parvaizqazi at my-deja.com parvaizqazi at my-deja.com
Fri Sep 1 09:43:56 EST 2000


In article <39AF6D3A.78999255 at immv.unizh.ch>,
  rogerrabbit at rzu-mailhost.unizh.ch wrote:
>
>
> parvaizqazi at my-deja.com wrote:
>
> > Recently I raised a mutant by site directed homologous recombination
> > strayegy in a wild strain of an Erwinia species with the help its
own
> > chromosomal DNA fragment ligated to a vector pRA90(bearing
> > chloromphenicol resistance)and later transformed to the wild strain
>
> I assume this is a ts or suicide plasmid?
>
> > To locate the vector insertion site we digested the total
> > DNA of the mutant with different restriction enzymes and
subsequently
> > hybridised with the labelled pRA90 as a probe but we could not get
very
> > clear signals of hybridisation sites although it was repeated many
> > times.
>
> How did you label your probe ? DIG? Radioactive?
>
> How did you detect it?
>
> Was the digestion complete?
>
> you could go to the methods-reagents newsgroup to get more help, but
> please describe more accurately what you did.
>
> Susanne
> Dear sir
I labelled the vector(pRA90)with dig-oxygenine(DIG) and digested the
Total DNA of the strain completely.I performed the gel electrophoresis
of the digested DNA and made a southern blot using standard nylon
membrane procured from Boehringer Manheim.I have doing such types of
DNA-DNA hybridisations frequently using DIG labelled probes and getting
good results but this is the first case where I encountered such type
of problem.Please suggest what to do next.Further I want to know
clearly what u mean if it is a suicedal vector.
yours
parvaiz
>


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