pBR322 with Eco RI - tet resistance?

Bill Melchior wmelchior at ntet.nctr.fda.gov
Wed Oct 27 10:03:17 EST 1993


In article <andres.43.751502550 at calvin.jci.tju.edu>, 
andres at calvin.jci.tju.edu (Andres Ferber) writes:
> I am trying to eliminate the HindIII site of PBR322 by filling the HindIII
> 5'overhang and then relegate the obtained blunt end.There is therefore a
> "gain" of 4 bases.Does anybody know if this will eliminate the TET resistance
> from the plasmid.
> So far I did not get any clones with TET and if a select in AMP I get some
> clones but they still have the HindIII site.

Two points:

Filling and religating a HindIII site should destroy the HindIII site,
suggesting that your filling reaction is not working. 

A year or so back there was a discussion here of the fact that, at least
with pBR322, tet selection (at the transformation step) does not work very
well.  I don't recall that any reason was agreed upon, but I don't think
you can rely on a failure to see tet resistant colonies after 
transformation as an indication that tet resistance has been eliminated, 
especially if you're not getting lots of amp resistant colonies.  What you 
need to do is transform and select on amp, then test transformants on tet.

(If your filling reaction is working partly, how about religating and then 
digesting again with HindIII to linearize plasmids that were not filled, 
which would make them much less efficient intransformation?  But this will 
work only if a reasonable fraction fo the sites ARE being filled.)
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