I am currently running a plasmid isolation. I am using a bacterial stab to grow the plasmids. We have used this stab a few times now. Unfortunately, we have not had the chance to make glycerol stocks of these plasmids. I streaked both an LB agar and LB agar with ampicillin plate. I left them in the incubator overnight at 37 degrees C. I do not see any growth or colonies. I will leave it overnight again in the incubator. I doubt that this will work because I have done this once before. If you have any suggestions please let me know. Thank you!!
If I understood correctly, your plasmid carries ampicillin resistance. If your bacteria does not grow, this probably means that your bacteria has lost the plasmid.
It is especially important not to extend the incubation, because there is a risk of the appearance of clones which are false positive, due to the disappearance of the ampicillin in the medium following the extension of the incubation.
Hi Sofiane! Thank you for the helpful information.
Sofiane Benyamina
To achieve successful bacterial plasmid growth, follow these key steps:
By optimizing each step and maintaining sterility, proper controls, and troubleshooting, you can improve the likelihood of successful bacterial plasmid growth and obtain high-quality DNA for your research needs.
I agree with Sofiane Benyamina as this has happened very often in my lab. What is most likely is that the plasmid has not completely closed, as even plasmids closed on themselves still create colonies.
I advise you check the process of plasmid formation (like digestion and ligation) or the ratio of lenght between insert and open plasmid. Also the lenght of the insert and its sequence can lead to a faulty ligation, since the insert bends onto itself.
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