4 replies to this topic

[microphobe]

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

[little mouse]

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

[microphobe]

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.

Edited by microphobe, 10 February 2009 - 07:07 AM.

[little mouse]

.. 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

[perneseblue]

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.