Nested Deletion paradox

Paul A Bucciaglia bucc0003 at gold.tc.umn.edu
Thu Apr 21 21:10:14 EST 1994


about the problem of nested deletions which look good on a gel but
when transformed into e. coli and miniprepped give widely diiferent sizes
than that shown on the gel:

I encountered the same problem.  To reduce the number of minipreps try
running your deleted time points on a 0.6 or 0.7% agarose gel, running the
gel as normal then excising the deleted bands and purifying them with your
favorite protocol (i use a simple spin column of the fragment over glass
wool; will give more details if anyone wants them).  This greatly reduces
the size range of the clones.  Note that the trade off is you need to
carefully watch your Exo III digestion times/temperatures so that you get
deletions about 200 bp apart which for me is an ideal length to maximize
new sequence on a gel but still give overlaps.

good luck and happy seq'ng.

paul bucciaglia




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