Since a few months ago, we have been occasionally getting colonies on control plates when transforming with kanamycin-resistance coding plasmids. This happens with different E coli strains. A possibility could be a lysogenic phage that codes for kanamycin resistance - is this likely? Could there be another origin for this contamination? We checked different sources of kanamycin, so that does not appear to be the cause. For the moment, we are carefully throwing away all experiments when this happens and doing these controls in every experiment - is there any other action that may be useful to take?
If your lab is routinely working with KanR plasmids, maybe one of your reagents (for example, your water used to make the kanamycin stock) has been contaminated with one of these plasmids. Also, if your kanamycin stock is too dilute, you may observe this (the kanamycin on your plates will degrade with time or if you add it to the LB when the medium is too hot). I would suggest remaking your antibiotic stock with molecular biology grade water, sterile filter it, and perhaps increasing the amount of kanamycin on your plates.
I beleive that it is more likely that you are dealing with a contaminant; rather than a temperate transducing phage. I have seen morphologically very similar colonies of plates, which subsequently proved not to be E.coli.
Correction to my previous post: contamination with KanR bacteria (likely harboring a lab stock plasmid) rather than free plasmid
what would be a quick test to see if a certain colony is E coli?
PCR with E coli specific primers comes to mind, but I've never done that
If you are dealing with wild type (lac+) cells I would streak the culture on a MacConkey agar plate - E..coli will give red colonies; Gram-positive contaminants probably won't grow; and Pseudomonas will give white/colourless colonies. Good luck
About the possibility of having contaminated kanamycin stocks, the problem has arisen with different kanamycin stocks prepared from different bottles and using different water.
Mark- another approach is to run a miniprep to isolate prospective plasmid from your resistant colonies. If you get nothing, then you have a contaminating strain of Kan-resistant microbe. If you recover plasmid, then you know that it is likely a lab generated strain that is mucking things up. Often times, these can sneak in from contaminated SOC medium, transformation tubes, or from whatever tool you use to spread the transformants on your plate.
we also receive now and then KmR background when working with Yersinia and I have been thinking these are spontaneous KmR colonies that arise especially if you grow the transformants for several days (sometimes, in allelic exchange experiments, we need longer times, 2-5 days, to get transconjugants). In such cases raising the Km concentration usually helps.
Additionally to what the colleagues say: Kanamycin is a bacteriostatic, more than a real antibiotic. Be careful if colonies grow slowly in the plates, I don't trust 2 days growth.
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