High concentration of antibiotics in amplification of plasmids - concerns about mutation of inserts in plasmid (May/01/2008 )

Hi, I am the newcomer and I am worried about what I have done with amplification of plasmids by transfoming competent bacteria such as DH5 alpha with high concentration of antibiotics, such as Ampicillin. People usually use 100ug/ml of Amp in LB broth, however I used 200-300ug/ml of Amp in LB broth. Right now I am concerned with whether under high selection pressure, the cDNA of interest cloned in plasmids may be mutated when plasmids are amplified in bacteria or not. Any explanation will be highly appreciated.


Philly

Almost certainly not. High copy plasmids are remarkably resistant to ampicillin. They hardly noticed.