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.The
transformants were selected on chloromphenicol based medium.This mutant
is blocked somewhere in the sorbitol metabolising pathway hence is
unable to utilize sorbitol and convert it to the particular ketoacid
which we use to check by acid zone formation and by HPLC as well in the
wild organism.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.Can you please suggest us something worth to get rid of the
problem.May I thank you in advance!
Parvaizqazi
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