2 replies to this topic

[jen_23]

Hey All,

Wondering if anyone can help.

I'm trying to transform clinical staph isolates with a lux plasmid and I am finding it impossible.
I have tried procedures using heat shock, cold shock, casein hydrolysate.... in duplicate and nothing.
I use chloramphenicol on BSA plates after electroporation and I usually get a 'film' of contamination.. so I went from 6ug to 20ug on plates and I either get nothing or contamination....

(Also I've tried this with 8 different starins...)

Has anyone successfully transformed clinical staph isolates before or knpw of a procedure which may be useful?

Thanks!

[phage434]

You may have restriction enzymes active in the strain, cutting your DNA.  You could test this by bead-beating some cells to open them up and mixing the cell mush with your vector to see if it is being cut.  Use RE buffer 2 or 4 as a good guess for the buffer needed.

[medivh]

i have been transforming s. carnosus which tortured me for a long time.
protoplast transformation is totally untrustable while electroporation works well. preparing competent cells is very important, so is the concentration and valume of DNA.
this paper might help well: http://www.biomedexp..._model_organism

jen_23, on Jun 4 2009, 04:58 PM, said:

Hey All,

Wondering if anyone can help.

I'm trying to transform clinical staph isolates with a lux plasmid and I am finding it impossible.
I have tried procedures using heat shock, cold shock, casein hydrolysate.... in duplicate and nothing.
I use chloramphenicol on BSA plates after electroporation and I usually get a 'film' of contamination.. so I went from 6ug to 20ug on plates and I either get nothing or contamination....

(Also I've tried this with 8 different starins...)

Has anyone successfully transformed clinical staph isolates before or knpw of a procedure which may be useful?

Thanks!