John and Alex
pulitzer at plauto.csata.it
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.
U. of Naples
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