I inoculated 20 mL of Rosetta pre-culture into 1L LB broth supplemented with 1 mL of Kanamycin and 1 mL of Chloramphenicol. After one hour of incubation, I checked the OD, and the value observed was 0.31; one hour later, it was 0.35, and after two hours, it was 0.32. To induce protein expression, I need a minimum OD of 0.8. Why cells do not achieve the desired OD? What could be the underlying cause for this lack of growth?
There are a few possibilities. First what concentration of antibiotics are you adding, to say that you add 1ml is meaningless without knowing the concentration of the stock solution. So it could be that you are adding one of the antibiotics at too high a concentration.
It could also be that you have lost or are rapidly losing one of the plasmids. You can test this by testing your strain with each antibiotic alone to see if they grow in culture.
Lastly it could be that your insert is somewhat toxic and you are just getting very slow growth due to that.
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