E. Coli Transformation

Kim Kirby kkirby at uoguelph.ca
Mon Oct 23 14:50:51 EST 1995


Stephen J. Palmieri (rocco at mail.utexas.edu) wrote:
:       I've got the weirdest problem known to mankind concerning my
: transformation.  I've got some sort of bacterial contamination that will
: not go away.  If  I plate untransformed cells(doesnot matter which strain)
: on an LB/Amp plate, nothing grows. But if I transform a ligation reaction
: I get these colonies containing no plasmid DNA and have a really bizzare
: odor and look in liquid culture.  If I transform a covalently closed
: plasmid about 80-90% of the colonies contain plasmid DNA so it is not a
: matter of degredation in my minipreps.  I've been using XL1-Blue and SURE
: cells because they grow very slow and I can tell a true transformant from
: a contaminating colony.  I've sterilized everything I could think of
: including changing my source of ddH2O  and increasing the Amp
: concentration to 100ug/ml.  Can someone out there please tell me what the
: hell is going on?  Anything would be much appreciated.

:                            Steve Palmieri
Hi Steve

I had the same problem last year.  My contaminating colonies smelled 
awful and they kept growing in the fridge.  I could not get rid of the 
problem but I found that I could work around it by picking colonies that 
did not grow in the fridge (much to my coworkers disgust since the smell 
was enough to knock you off your feet).  The problem just went away on 
its own after about 2-3 months. None of the lines I made during that time 
show any sign of the problem now.  

Good Luck
Kim



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