John and Alex pulitzer at
Thu Mar 16 13:24:38 EST 1995

Hi! In constructing a vector, a few steps a go, I removed an unwanted
EcoRI site from a plasmid by cutting, filling in with Klenow, and 
religating. I find now that as expected there is no "true" cutting at
this site however I note two unexpected occurences.(A) this plasmid,
but not others devoid of EcoRI sites, get nicked in the presence of
EcoRI and, (B) although at lower efficency cut, especially under
 semi-* conditions (enzyme a bit high, hours of incubation, etc). 
I judge cutting at this site by the fragment length I get cutting
with a second enzyme. Actually, I always get at least some cleavage 
even when enzyme is dilute.

Could it be that the residual EcoRI sensitivity is due to the
two adjacent EcoR* sites created by filling and religating an EcoR1
site? Any ideas, observations? Thanks. John

PS I am not sure how this posting is going to read. I am using the 
Netscape News posting add-on and text is not wrapping around in the 
window, so I am hitting a lot of RETURNS. Apologies if this comes out
a mess.                                                                             a mess.   

John Pulitzer
Dept.of Genetics
U. of Naples

More information about the Methods mailing list