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