ExoIII Deletions of Long Inserts???

mmelan at marcie.wellesley.edu mmelan at marcie.wellesley.edu
Fri Nov 12 18:16:07 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

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