gt11 blue/white plaques?

Warren Gallin gal-1 at bones
Fri Jan 27 17:07:30 EST 1995


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On Fri, 27 Jan 1995, Jim Mensch wrote:

> I have a lambda gt11 cDNA library which I obtained from ATCC.  In the
> course of titering the library, I added X-gal and IPTG to the top agar
> of one plating, expecting to see a large majority of white (colorless)
> plaques indicating phage carring an insert in the lacZ portion of the
> gt11 construct.  In fact, I saw about 80% (eyeball estimate, too many
> plaques to count) blue:20% clear.  Can anyone confirm my speculation 
> that since the insert (EcoRI) site is near the 3' end of the lacZ
> coding region many inserts do not interfere with the complementation
> of this lacZ product with the defective host B-galactosidase?

gt11 is not designed for alpha complemetation.  As you note,  the cloning 
site is at the far C terminus of an intact beta gal gene.  If you are 
using a complementation host like XL1-Blue instead of the appropriate 
strains (Y1088, Y1089 or Y1090, I forget which off the top of my head) 
then you will complement whether or not there is an insert because the 
insert will not disrupt the alpha peptide portion of the molecule.  The 
mystery that you have to figure out in that case is why you have any 
clear plaques.  If, on the other hand you have the host cell, then the 
problem could be that the library has been re-amplified.  The 
non-recombinant phage will outgrow the recombinant, so amplifying more 
than the first amplification when the library is first made will lead to 
progressively high proportions of non-recombinant phage.

Regards,
Warren Gallin 



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