E. coli not growing in liquid cultures :( - (Aug/27/2010 )

Hi Bioforumers,

I have a ligated mix from restriction digests which I transform into XL2 Blue cells. I select colonies by blue-white color screening on LB-Amp plates containing X-Gal and IPTG. At this point I assume that my ligation product would be found in the white colonies so I pick a few and I inoculate 5mL LB-Amp cultures. Incubation takes place at 37C O/N. The problem is the white clones are not growing in the liquid culture whereas the blue clones (negative control) are growing!

I have been trying to obtain my DNA from the white clones for the past week but my starter cultures are not growing!

Can someone please advise and give any suggestions??

Many Thanks

Are the white colonies you're picking all in close proximity to blue colonies? Perhaps they're satellite colonies, and thus are not really transformants (and thus are not resistant to ampicillin because they don't actually contain the plasmid). Pick all the white colonies using sterile toothpicks or pipette tips and patch them to a fresh L-amp plate; see if they grow.

If u incubate for more days satelite colonies will appear. pick 24 hrs white colonies and patch xgal, iptg antibiotic plates with sterile tooth picks.

So yesterday I decided to pick some white clones and streak them on fresh LB-Amp plates. Incubated O/N at 37C. Result: NO COLONIES!

A colleague of mine suggested to try transforming into XL10 cells instead of XL2 Blue's.

Does anyone have any experience with these?

Thanks for your suggestions guys

Your problem is unlikely to be due to the recipient strain used. More probably you're incubating your transformant plates too long, allowing satellite colonies (which will be white) to appear around the blue colonies (assuming there were blue colonies on your plates), or the Amp concentration in your transformation plates is too low. Streak some of your untransformed reciepient strain to your Amp plates -- they should not grow. Another possibility is that you forgot to add X-gal to your transformation plates; thus all colonies will be white.