Help???How to cip treatment with blunt-end vector???

Dom Spinella dspinella at chugaibio.com
Thu Aug 21 15:22:03 EST 1997


C. Rodemer (in a triumph of modesty and taste) writes:
> I think many things in the Maniatis book are unnecessary,old and time
> -consuming. 
> 
Well, you can get away with cutting corners once in a while, but sooner
or later it comes back to bite you. No doubt your own forthcoming recipe
compilation will be far superior.
> 
> I cut my PBluescript with EcoRI and didnt precipitate anything.I just
> added buffer for CIP and some Units CIP .Then incubate at 37C. 
> 
You're a cloning stud!  However, since the original poster didn't say
what restriction enzyme he was using for blunting, I thought it best to
send him the "long" procedure since Cip isn't always fully active in all
restriction enzyme buffers.
> 
> Well, I just made two phenol/chlorof/IAA extractions and precipitated the
> vector with 2 vol ethanol. Then I used it for ligation. 
> Obviously it worked. I screened only 10 cols and I found the recombinant
> pBS in one colony.
> 
> bye 
> C.R.

Way to go, ace! Looks like next year's Nobel is a lock for ya.

Later much
D.S.



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