blue white selection
mlsulliv at wisc.edu
Thu Jan 26 12:35:26 EST 2006
My guess is that it wouldn't work. Not sure, but your agro strain
might already be positive for beta galactosidase (everything blue).
Otherwise, the color selection for most plasmids is based on alpha
complimentation, ie., there is a small fragment of lacZ encoded on
the plasmid that compliments (in trans) an incomplete lacZ fragment
encoded by the host bacteria. Without the host-encoded part, the
plasmid encoded lacZ fragment doesn't have enzyme activity. Also, if
you made a construct in a plasmid with color selection, usually
you're going to white (lacZ-) colonies, so I'm not sure how color
selection would help in identifying agro transformants. Usually the
purpose of color selection is to identify recombinant plasmids when
you're building your construct in E. coli.
But, I would presume you transforming a construct you already built
in E. coli into agro and using antibiotic resistance to select
transformants. If so, I'd think that most any agro colony you recover
is going to have plasmid in it. I usually just do minipreps of a few
colonies (Qiagen actually works quite well for agro) and check for
plasmid by restriction digest. Sometimes the DNA from Agro is of poor
quality and low yield so digests don't work well, in which case I
simply retransform the agro plasmid prep back into E. coli, and
analyze that to make sure my plasmid made it into agro intact and
Hope this helps.
On Jan 25, 2006, at 8:18 PM, newsnet customer wrote:
> Does blue white selection work with Agrobacterium strain AGL1?
> trying to screen transformed colonies without using PCR.
> Sharp Tool
> Methods mailing list
> Methods at net.bio.net
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)
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