Cloning small DNA fragments. Bacteria are not growing - (Jul/25/2007 )

Hi guys,

I am having huge problem with the cloning of small DNA fragments (that corresponds to microRNA) in E. Coli .
I've transformed the bacteria with pGEMteasy and I obtained positive white colonies. However, these colonies are very small (comparing to the blue ones) and they don't grow on liquid LB .
If anybody knows what the hell is happening?? Any solution to grow these bacteria in liquid (I'd really like to do some miniprep to sequence)..

Thank's in advance


Steph

i don't see size differences with negative and positive clones of pGEMT. I would suspect you got some contaminants that behave like satellites.

as Fred_33 stated, quite likely the "white" colonies are satelite colonies, colonies which are empty but due to degradation of ampicilin by secreted beta-lactamase from proper transformants (probably the blue colonies), have managed to grow.

Not your lucky day.

As others claimed, probably satellite colonies. What is the concentration of ampicillin is in your plates?

I use Ampicilin 100µg/ml
Well, I don't think it's satellitte, cause I checked them by PCR and the pGEMT easy is inside!!!!
And I tranfered it on new LB-Agar-Ampicilin plates and they growed....

and these transfered colonies don't grow in liquid LB?
honnestly, a 50µg/ml amp solution is sufficent for getting good selection. In the liquid medium, bacterias are more accessible to ampicillin than in plates. So try to decrease your liquid-ampicilin concentration and tell us