Multiple colony types from streaking 1 E.coli colony - (Feb/10/2009 )

Hi all

I have some really puzzling results when I tried to transform pGEM-3z into DH5-alpha E. coli.

I transformed 1ul of pGEM-3z (all that we had left!) into DH5-alpha and spread it on LB-agar with ampicillin.

The next day there were clearly 2 colony types - a few big and many small (at least 100x more small). I miniprepped plasmid DNA from both and the small colony grew very slowly in liquid and gave much less DNA but the plasmid was correct in both cases.

I wanted to check that it would be OK to do blue/white screening so I streaked 2 of each colony type onto LB-agar + ampicillin with IPTG and X-gal.

The big colonies gave rise to BOTH big/white and small/blue colonies (~100x more small than big).

The small colonies also gave rise to both colony types (~1000x more small than big), but there were about 3 big colonies which were also kind of blue. These big blue colonies also contained the correct plasmid.

I have no idea what is going on here, please let me know if you have seen this before! I need to be sure that white colonies result from insertional inactivation later on when I use the pGEM3z for cloning, so having white colonies with the empty vector is not good.

MP

I think you have a contamination with non E coli bacteria, (that were also transformed ?!)

I thought that too until I streaked out the individual colonies and I still couldn't get a homogenous population... unless the contaminant and the Coli have some sort of weird symbiotic relationship and form mixed colonies or something?

Also, I was very careful not to touch surrounding colonies when I was picking the colonies for secondary streaking.

.. And it doesn't seem to be sattelite colonies neither (non transformed bacteria that starts to grow when the antibiotic is consumed).
It's strange

could the plasmid transformation be contaminated? Ie you have two types of plasmid, one which has the functional lacZ gene (pGEM-3z) which give a blue colouration. And the other is a plasmid where the lacZ gene has been rendered non functional.. ie it has a mutation (or gene insertion.)

I once experience something similar but not on the scale you are experiencing. Upon transformation of my empty vector into e coli with X-gal, i found a few white colonies... when all should be blue. One of these white colonies (there were only about 10 when I have 100s, maybe 1000s of colonies that were blue), contained a plasmid that was being worked on in the lab. I assumes it was a contamination problem, more so as my lab reused electroporation cuvettes.

Hi!, I have the same problem and I don't know what to do. I tried to isolate a positive colony, get plasmid of it and transform, but the two populations appear again.

How do you solved the problem?