ExoIII Deletions of Long Inserts???
niflab at biology.ucsc.edu
Mon Nov 15 16:07:59 EST 1993
> 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
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.
Robert Kuhn, PhD
University of California
Santa Cruz, CA 95064 USA
email: rkuhn at orchid.ucsc.edu
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