Uni vs. bidirectional Exo III deletions

ww40 William_D_WARREN at UMAIL.UMD.EDU
Wed Aug 19 18:18:00 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
[stuff deleted] 
>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

Jim...
I suspect that your problem is that you are using too little
of the Exo III - because unless there is an excess of enzyme you
won't get a set of uniform deletions. For UNI-directional deletions
I typically use 100 U of Exo III per ug of DNA (typically 4-8kb). Also
the rate of digestion is generally linear with temperature, progressing at
around 300bp/min at 35oC, 500bp/min at 39oC. And although I have never
set out to make BI-directional deletions myself, I have seen some
when co-workers have forgotten to perform the second digestion to
produce a protestive 5'overhang, so I can assure you that IT DOES WORK.

Another mistake I've seen people make is to add 10xExoIII buffer
(which in my protocol contains 7mM MgCl2) to their DNA that has been
suspended in TE - this results in NO DIGESTION AT ALL since the Mg2+
gets all sucked up by the EDTA. Either resuspend the precipitated DNA
in 1xBuffer or in H2O then add the 10xBuffer.

I have had troubles when trying to generate deletions that have
alot of secondary structure. Do you know the sequence of your DNA ?

Hope this helps

Bill Warren
Center for Agricultural Biotech
University of Maryland

(ps "DNA Dipstick" gets my vote for "Most Useless Kit of the Year" too!)




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