Kanamaycin bugs grow on Chloramphenicol plates... - (May/19/2010 )

Hi there,

I recently found that my Kanamycin resistant bacteria grow on Chloramphenicol plates (but not on Ampicillin plates). I have used Chloramphenicol concentration of up to 35ug/ml, made fresh plates (not too hot), made new Chloramphenicol stock from powder, picked single Kan resistant colonies and made miniprep of single Chloramphenicol resistant bugs (no insert). My Kan resistant vector is peCFP-N1, Chlor resistant is pBAD33-CyPet. Bugs are homemade DH5a.

What can I do? Any suggestions welcome!


Cheers,

Minna

We've seen "breakthrough" of Cm-sensitive E. coli at 34 ug/ml on L-agar; we now routinely use Cm at 50 ug/ml to select for Cm-resistance. ATCC recommends a range of 25 to 170 ug/ml to select resistant E. coli (see here).


I also seem to recall reading somewhere that the activity of chloramphenicol actyltransferase is sensitive to the pH of the media...

Thanks for your answer. I will try increasing the concentration of CAM, one batch of 50ug/ml, one with 150ug/ml. That should kill those critters!
pBAD is a low-copy vector, does CAM concentration have any effect on that? I mean, does increasing CAM concentration result in selection of high CAT expressing cells (or lower number of colonies in my case)?
What do you mean with the pH of the LB medium? I never measured that...

Cheers,


Minna.