No Blue Colonies...I'm not that good.
seraln at _NO_SPAM_netscape.net
Tue Feb 19 04:49:55 EST 2002
I would say that the bacteria you're using is not alpha-complementable, it's
just a lacZ- strain.
Vectors like pUC19 don't carry the complete LacZ gene, just a truncated form of
it, and it only works within microorganism which carry the other part into their
chromosome. If you need the blue/white selection, choose a strain with the
phi-80 lacZ(delta)M15 mutation.
Hope this helps...
> I'm trying to shotgun clone in chromosomal fragments of a cellulolytic anaerobe
> into pUC19. Everthing has gone well, and I'm using a Rec- strain of E. coli
> for transformation. But all I get are white colonies. NO blue colonies at all,
> not even on the control bacteria with unligated plasmid. Some plates had
> X-gal, and some had Bluo-gal. Some had IPTG and some did not. Amp
> concentration was 10mg/ml, and I did 1:1, 1:10, 1:100, and even 1:1000
> dilutions. Colonies were in numbers in proportion to the dilutions, so they
> were my E. coli. Why didn't any of them turn blue?? I don't necessarily need
> the b/w colony selection for what I'm screening for, but it would be helpful.
> I would appreciate any help. Thank you, S. Polek
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