Nru I digestion problems

Gary K. gtk10583 at glaxo.com
Fri Feb 23 14:15:49 EST 1996


In article <tyr-2-2102961552580001 at duckter.glaxo.med.ualberta.ca>,
tyr-2 at bones.biochem.ualberta.ca (Karl Fischer) wrote:

> Hello,
> 
> We are having problems digesting one of our plasmids with the restriction
> enzyme Nru I. The plasmid was propogated in E. coli DH5alpha, purified on
> a Quiagen column, and evaluated by UV spec (260/280 ratio 1.9). Five
> micrograms was digested O/N at 37 degrees with 20 U in a final volume of
> 20 ul then qs'd to 40 ul and further digested with an additional 20U of
> enzyme. Gel analysis shows no digestion.
> 
> Has anyone experienced wonky cutting with this enzyme? We are currently
> sequencing the region where the Nru site lies, though the DNA containing
> the site is from a prototype clone (ie. fully sequenced by another
> laboratory).
> 
> Any information is greatly appreciated.


Karl,

Here is your problem:  Nru I activity is blocked by overlapping dam
methylation.  The strain of bacteria you are using, DH5 alpha, is dam+. 
What you need to do is transform your plasmid into a dam- strain such as
DM1.  The lack of dam methylation will cure the problem.

Gary



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