The missing plasmid…

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    • #8085
      SororSaudade
      Participant

      Hello all!

      I was trying for ages to transform some Agrobacterium cells and never manage to get any colonies. When I finally got one colony on the transformation plate I did a PCR on it and it amplified very well a fragment the size I was expecting.
      After this I grew these bacteria on solid and liquid medium with the proper antibiotics and they grew very well. When I did a new PCR (with negative and positive controls) in these new cultures I could never get any kind of amplification.
      I thought the antibiotic might be damaged and did a new one, but got the same kind of results (even with higher concentrations of it).

      Has this ever happened to anybody? Can bacteria get resistant to some antibiotic without the resistance gene?

      If some one could help be with this, I would really appreciate it. 🙂

    • #75195
      canalon
      Participant

      Spontaneous resistance is not impossible. It depends on the antibiotic though. I am not familiar with Agrobacterium though, so it is impossible to judge how frequent it is.

      Which antibiotic are you using?

    • #75200
      SororSaudade
      Participant

      I’m using kanamycin.

      Agrobacterium are naturally resistant to rifampycin… and I also add this to the medium.

      But can you explain me how do they get this spontaneus resistance?

      Thanks 🙂

    • #75208
      canalon
      Participant

      I was not sure for kanamycine, but if you look up the mode of acion you will see that it binds the ribosome and prevents elongation. Like many aminoglycoside with similar mode of action (and rifampicine, which is not an aminoglycoside, but also blocks the ribosome) resistance can be acquired by some mutations in the target which will keep the function intact.

      With E. coli this kind of mutation appear in 1 in 10^9 or 10^10 bacteria, but in case of selection that might be enough for you to have picked the wrong one. as for the false positive for the first PCR, I suppose you had a negative control… but did you re-isolate the colony before doing the PCR? Otherwise remeber that you use quite a lot of your PCR target to make your transformation, and you need very little to get a good amplification…

    • #75213
      SororSaudade
      Participant

      wow… thanks for the explanation 🙂

      I already knew I have to do it all over again… just wanted to know what might have happened

      Thank you once again

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