Hello Science community,
I am working with several Agrobacterium strains (GV3101 pMP90, GV3101 pMP90 & EHA105). All of these strains are sensitive to the expected antibiotics when using only glycerol stocks of the strains for inoculation of solid and liquid media. However, after I made them chemically competent for transformation, the selection of the strains no longer works.
I have repeated the competenting with freshly made solutions to rule out contamination and changed the antibiotics used, but the problem remains the same.
Perhaps someone of you can give me an explanation?
Virginia Zahn The only logical explanation - there is already a number of multi-resistant Agrobacterium in your stocks, and for some reason they are more viable in the process of preparation of competent cells. You can re-isolate a single colony susceptible to antibiotics and repeat the transformation.
Yes, I checked them and they worked. Nevertheless, I also replaced them with newly purchased ones as a precaution.
Are you starting from single colonies that you streak from the glycerol stocks? It might be you have a low level of resistant contaminants in the glycerol stock so when you test it most the colonies are sensitive but when you do a transformation the low percent of resistant cells will grow.
I inoculate the culture directly from glycerol stocks and do not use single colonies.
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