mutagenesis kit

Robert J. Nelson Robert.Nelson at ed.ac.uk
Wed Sep 5 12:27:01 EST 2001


Hi Wolfgang,

I've never been able to explain it satisfactorily, but it does seem to be
consistent in that it is the whole of the mutagenic region that is
duplicated. 
  
   -------->-------------------------------------------
            -------------------------------------------<--------

   -------->------------------------------------------- -------->
  <-------- -------------------------------------------<--------

One idea is that if the mutagenic product comes apart at the nicks it leaves
overhangs that unincorporated primers could anneal to.  Whether this could
produce a viable plasmid by ligating the blunt ends I don't know.  Or maybe
if the ends are chewed back a bit by the 3' to 5' exonuclease activity of
the Pfu...  

Just grasping at straws I think.  If anyone else has any ideas...

In some of my mutagenesis reactions I include a silent restriction site to
speed up the analysis of clones, so it does throw up the possibility of
errors if you screen solely by restriction digest.

bob


---
> Dear Bob, 
> 
> I also observed this sometimes. Any ideas why? Sort of
> incorporated primer dimer?
> 
> Wo
> 
>> ... and occasionally a
>> plasmid with the mutagenic region duplicated.
> 

---




More information about the Methods mailing list