Initially, I did Agrobacterium transformation with my constructs and gave many colonies. Then did PCR and sequencing for several colonies and showed that my constructs were transformed. Then I re streak my colony to another agro plates for back up. After one week, I restreak again the agro cells to another plate (same conditions and everything) to be used for rice transformation, however after 3 days it did not grow at all? Has anyone have an idea of what happened? I am assuming that there is no contamination happened there...
1. Normally, after 3 days, you should be able to see tiny colonies (at least). But, probably give it another 1 or 2 days to see bigger colonies.
2. Double-check the plates and make sure that they have the right antibiotics; sometimes different plates may mix-up (although you said it is same condition)
3. Do you keep liquid bacterial stock (from positive colonies) at -80C? You can re-streak from it.
4. When you re-streak, make sure the iron inoculation loop is not burning hot (from flame sterilization) and kill the bacteria.
Are you storing the Agrobacterium plate in a refrigerator at 4 degree Celsius. Once the bacteria grow (approx. 3 days at 28 degree C) the plate should be stored in a refrigerator.
Possible reasons for the described problems with Agrobacterium:
bacteria were kept too close to freezing element in refrigerator and might be occasionally frozen;
antibiotic concentration was occasionally increased during medium preparation;
too hot inoculation loop (as it was mentioned by Yuan-Yeu Yau in the first comment)
Try to repeat the inoculation; if it fails again I would advise you to perform new transformation. As well, you can scratch the whole bacterial material from your Petry dish and inoculate it into liquid medium. If you have there any live cell it will grow. But I would advise to prepare new strain. I support Jelli Venkatesh that restriction analysis of plasmid is better for transformation confirmation.
Yuriy suggestion of scratching the whole bacterial material from your Petri dish and inoculating into fresh liquid medium might work and results in higher plant transformation frequency based on my experience.
Dear Cantos
I too agree with Narendran sir , once your construct was confirmed you can inoculate single colony and plate the same thing next day. and store the plate in refrigerator and you have to revive atleast for each 15 days , so there will be no contamination.
and at the same time as you are mention there was a no growth , that means unknowingly u pick up some satellite colinies or else your antibiotic is more.
Thank you
Thanks folks for the recommendations and suggestions. I found out just yesterday, that our incubator's temperature changed from 28C to 37C which I think affect the growth of the bacteria, I made another re-streak from my glycerol stock and worked well! I still appreciate the comments and response. Many thanks!
@Christian,
It is good to know that you found the problem. This also happened in my old lab when there was only one incubator and several people needed to use it for different researches. Some people used it for E. Coli culture (37oC) and some went for Agrobacterium culture (28oC). So, eventually we decided to put a note on the incubator to warn other users whenever the setting of the incubator is changed.
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