Uni vs. bidirectional Exo III deletions

the End jgraham at bronze.ucs.indiana.edu
Wed Aug 19 13:39:01 EST 1992


Hello networkers,

I recently was rather successful in creating some deletions in an
Bluescript based plasmid using the Exonuclease III/ Mung Bean
procedure as described by Strategene in their literature that
accompanies the Bluescript vector. The problem is that I would like to
create BIdirectional deletions centered at a unique site and the
twenty or so clones I have sequenced all have deletions extending in
either one or the other direction from the target restriction site.
This site generates the appropriate ends for Exo III deletion in both
directions.

I am using 30U of exonuclease for 2.5 micrograms (1 pmol) of
linearized plasmid DNA and digesting at room temperature for intervals
under 60 seconds.

Samples taken at 2 secs show a pattern of unidirectional deletion of
between 1 and 45 bp, while longer incubations do not show any
deleteion. These deletions in samples taken at the first time point
appear to be products of a step-wize removal of residues, as many
identical clones are seen that are deletions of either 12, 30, or 45
base-pairs. I have not detected any of intermediate size in the 75
clones I have looked at.

I have tried reducing the enzyme concentration and increasing the
reaction time, and reducing the temperature to 16 C and increasing the
enzyme concentration three fold, but have yet to analyze these
reaction products.


Has anyone successfully created deletions in both directions with Exo
III or experienced similar problems ?

Thanks much,

Jim
J. E. Graham
Biology/Chemistry Departments
Indiana University
.............."Il n'y a pas des sciences apliquees, mais il y a..............
.................des applications de la science". L. Pasteur.................



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