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Does kanR gene confer resistance to Ampicillin? - (Apr/27/2008 )

I transformed pET-28a (containing a KanR) into E. coli, plated all the transformed mixture onto LB plate with Ampicillin by mistake. The following day, I only saw 3 colonies on the plate while for the positive control, the efficiency of transformation was over 10^7/ug of DNA. I realised I put the transformed cells into the wrong antibiotic plate but I was curious how it could arise. I isolated the 3 colonies and did a plasmid prep, the isolated plasmid was only ~ 2.0 kb, while the pET28a is ~ 5.4 kb.

The possibility of cross contamination is ~ 0. Because I used new cuvette for the transformation and I did not have any plasmid smaller than 5 Kb.

Can anyone offer a possible explanation?



No, the Kan resistance cassettes (there are several different ones) can often convey resistance to Neomycin and Gentamicin, but not to ampicillin. Spontaneous mutants can arise, but more likely this is contamination from an amp resistant strain or plasmid. You could sequence it, if your wonder continues. 2Kb is small for most common plasmids.