Dear friends,
I have been facing a problem for the last month in the lab. We are using the gateway expression vector, pk7wgf2, to transform the agrobacterium strain EHA105. We added rifampicin (25 mg/L) and spectinomycin (100 mg/L, for selection of transformed cells) for selection. But only after overnight incubation was very robust growth found in the plates.
Please give me the possible reason where we are making the mistake.
There can be multiple reasons for the problem that you are facing. The first thing that you can do is check your competent cells on rifampicin as well as spectinomycin selection plate, if your empty strain is still giving colonies that means your comp cells are contaminated, and in that case, you should prepare the comp cells again with a fresh glycerol stock. Another thing that you can try is increase the rif antibiotic concentration to 50 µg/ml. Also, I am assuming that after transformation, you are incubating your plates at 28 to 30°C and the protocol that you are following for Agrobacterium transformation has been tested before.
Hope that helps.
I think you should use the fresh antibiotic and reduce the usage of Agrobacterium cell, if you transfer the entire plasmids into the Agrobacterium component cell.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering