Hello I have been trying to transform pet28b plasmids into Rosetta cells. I have tried two plasmids that already show expression in BL21 ( DE3) cells. Is there a list of compatible plasmids that can only be transformed into Rosetta cells? Also, does the factor that Rosetta cells have a resistant gene to Cloramphenicol has something to do? because my pet28b plasmid has a resistant gene to Kanamycin
Rosetta 2 (DE3) can host almost any plasmid except these with chloramphenicol-resistance, it is specifically engineered for pET vector-based expressions and is compatible with pET28b. Home made competent cells can lost their competence quickly, sometime you need to add more plasmid DNA to get a good number of transformed colonies.
pET vectors should be fine, but be careful with kanamycin, it is a protein synthesis inhibitor so too much will kill the cells, versus ampicillin which is bacteriostic so they can sit around and wait for it to degrade to a level they can deal with and then start growing.
Thank you very much. I will consider reducing the antibiotic concentration and increase plasmid concentration. Thank you again
To main the Rosetta 2 (DE3) strain, do we have to grow them in any antibiotic? Chloramphenicol?
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