URGENT-Selecting for transformed bacteria cells - (Mar/01/2006 )

Hi all,

I have a problem with the selection of my transformed bacteria.

The bacteria strain that I am working on has a gene knockout made, via insertion of a gentamycin resistance gene as such this bacteria has resistance to gentamycin at ard 50ug/ml.

The plasmid that I have to introduce into this bacteria strain has also to be selected using gentamycin.

I have tried to plate my bacteria on agar plates with different concentrations of gentamycin from 50ug/ml to 1000ug/ml and have done dilution in order to get single colonies but there is no transformed bacteria.

However when I repeat this entire transformation procedure with my wild type bacteria that has no instrinc resistance to gentamycin I could get my transformed bacteria.

Therefore I would like to know if anyone of you have meet with this problem and advices to this problem.

Any comments are welcomed.

Thank you so much.

hi
i thinkg that if you have 2 gentamicin resistance gene, that do not signify that your bacteria will resit 2x concentration.
Moreover, for your first problem, as the bacteriaitself resist to gentamycin, there is no need to get extra resistance gene. So the plasmid is lost...
you may have faced to transformation difficulties, but i don't belive explain all the first assay.