Hi,
I have rosetta (DE3) with incorporation of tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. I cloned a gene in kanamycin resistant vector.Later, I transformed and plated on LB Kan plate, Do you think that due to absence of chloramphenicol, it will through out plasmid?
If yes, how much chloramphenicol should be added?
Yes it will after many generations. You need to add 34ug/ml chloramphenicol in your media.
Good luck.
They will definitely dispense with the plasmid if there is no selection reinforcing it. Maintaining a plasmid, especially one that is going to force the cell to produce many copies of a protein it doesn't want or need, is an extreme use of energy that is highly unfavored.
DE3s are notorious for dispensing plasmids which is why you should transform and plate them fresh before any expression protocol, never use plates that are more than a couple days old, and never cryo or -80C preserve plasmids in DE3 (Top10 or DH5a are much better for this purpose).
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