Contamination of DH5 alpha stocks? - Colonies produced with negative control (Aug/13/2007 )

Good morning

I was hoping someone might have some suggestions for a recent problem we have been having in the lab. For bacterial transformations we produce our own chemically competent DH5 alpha cells and transform using heat shock. Our two most recent batches have produced colony growth on ampicillin plates in the negative control (heat shocked but in the absence of any DNA). The ampicillin is from a batch we have used previously with out any problems. I selected a single colony of untransformed DH5 alpha from an unsupplemented LB plate. I grew it in LB broth (no ampicillin) for 4 hours (in a falcon tube) and then plated an aliquot onto 1) unsupplemented LB agar 2) LB agar + ampicillin. On the first plate a lawn of bacteria grew as expected, the second plate contained 8 fair sized colonies. I assume we have contamination of some sort, has anyone else had any similar problems?

Thanks

Sally

is there any natural selectable marker in DH5alpha strains ?... that one should be used for counter selection.
if not, i guess you need to ask an other lab to kindly pass you some bacterias.

Hi Sally

How many colonies do you obtain when the transformation negative control is plated on ampicillin? If it is just a small number (compared to plating on LB) it is probably not a contamination, but just some clones/mutants that are less sensitive to the ampicillin.
The fact that you obtained a similar result in the second experiment, where you grew up a single clone and again obtained a small number of apparently resistant colonies would suggest this is the case. My suggestion would be to double the ampicillin concentration in the plate and see if you get better selection.


If you need any further help, let me know.

Nick.