blue white selection

Michael Sullivan 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  
without rearrangement.

Hope this helps.

Mike Sullivan

On Jan 25, 2006, at 8:18 PM, newsnet customer wrote:

> Hi
>
> Does blue white selection work with Agrobacterium strain AGL1?  
> trying to screen transformed colonies without using PCR.
>
> Cheers,
> Sharp Tool
> _______________________________________________
> Methods mailing list
> Methods at net.bio.net
> http://www.bio.net/biomail/listinfo/methods

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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