ExoIII Deletions of Long Inserts???

[Ludwig Lab-UCSC]

> 
> Dear Methodnetters,
> 
>         I am currently having a problem trying to do ExoIII deletions of a
> 5kb insert fragment in Bluescript.  I looks as though the ExoIII does a fine
> job of chewing halfway through the insert (i.e. reactions are beautiful reducingthe 8kb linear DNA down to about a 5kb fragment) but further reactions (later
> timepoints) show no DNA in the lanes.  I am starting with 3-4 ug of DNA in the
> reaction and using 300 units of enzyme.  Has anyone else had a similar problem?
> Am I not starting with enough DNA? Is there a S1 nuclease problem? Could this
> be some strange problem specific to this piece of DNA?  I am using the original
> Henikoff procedure.  Is there a better method?
>         Any help on this problem would be greatly appreciated...I'd love to
> get started sequencing soon.  Thanks,
> ===============================================================================
> Melissa Melan                           mmelan at lucy.wellesley.edu
> Department of Biological Sciences
> Wellelsey College
> Wellesley, MA  02181
> (617) 283-3137
> ===============================================================================
> 
> 

Melissa,
	Have you thought about doing "dropouts"?  Doesn't address the prob 
you relate, but it gives smaller insert to work through.  By dropout 
I mean cutting with an enzyme that cuts in the insert and in the poly-
linker, to release a fragment and recircularizing to bring the rest of the
clone next to the primers site.  It's very handy, and gives much better control
over deletion size.  Just a thought.  Good luck.
			--b0b kuhn

      ------------------------------------------------
	Robert Kuhn, PhD
	Sinsheimer Laboratories
	University of California
	Santa Cruz, CA 95064  USA
      
	email: rkuhn at orchid.ucsc.edu
      ================================================