cloning

LEIBOWITZ at OCELOT.RUTGERS.EDU LEIBOWITZ at OCELOT.RUTGERS.EDU
Tue Nov 26 09:16:06 EST 1996


In response to the query below, we have recently had trouble cloning a cDNA
derived from yeast in E. coli, and by using PCR of different regions of the
cDNA determined that any construct having a specific 700 base region seemed not
to clone in the polylinker of pUC18.  We found deletions, incorrect sequences,
and all sorts of garbage, but never anything that had our PCR primers (unlike
our experience with many other cDNAs.  So, we predicted that if, indeed, our
problem was due to toxicity of the cloned fragment, that  toxicity was likely
to be due to the expressed protein made as the alpha-fragment fusion product. 
So we repeated the experiment growing the E. coli on glucose and foregoing
blue-white selection, and we got our clone.  But we could not show that is was
actually toxic on X-gal plates.  So our solution was not intellectually
satisfying, in that toxicity could not be proven.  But, we did get our clone.

Sincerely,
Mike Leibowitz,
RW Johnson Medical School

>Hello everyone,

>I have a simple question to ask...how frequent/common is it for people to 
>experience deletions/rearrangements when they are cloning their favourite 
>gene?  I appreciate that the problem is likely to be strain-related and 
>I have tried several different strains, finding my plasmid to be more 
>stable in JM101 than any other.  But still, deletions occur.  We even 
>jest about "vanishing genes".  Our lab often discusses possible toxicity of
>some DNA sequences to E. coli and it has been suggested to us to try directly
>transforming into yeast.  

>I am interested to find out how often this sort of problem arises and how 
>other labs approach and overcome it.  

>Any thoughts would be appreciated.

>Cheers,
>Marguerite.

>Marguerite Evans
>School of Biochemistry and Molecular Genetics
>UNSW  Sydney  2052
>NSW
>Australia

>voice: (+61)(2)385 2030
>fax:   (+61)(2)313 6271
>email: p2158740 at acsusun.acsu.unsw.edu.au





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