Mutation and Sorbitol *******a

[Susanne Rohrer]

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