ExoIII Deletions of Long Inserts???

[mmelan at marcie.wellesley.edu]

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,
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Melissa Melan                           mmelan at lucy.wellesley.edu
Department of Biological Sciences
Wellelsey College
Wellesley, MA  02181
(617) 283-3137
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