Nested Deletion paradox

Casey M. Finnerty cmf5 at cornell.edu
Mon Apr 18 12:35:38 EST 1994


Dear Netters:

I've been sequencing a couple cDNAs using nested deletions (made with the
Promega Erase-A-Base kit) and have consistently run into something that
puzzles me. After digesting the DNA with Exo III, blunt ending with S1
followed by Klenow treatment, I will take aliquots of each time point in
the deletion series to analyze by gel, as recommended by the kit. In the
meantime, I set up ligations that I leave overnight at room temp. Assuming
the restriction digests I used to prepare the DNA worked well, I usually
get a very nice ladder of tight bands ranging in size from my cut vector
plus insert to the empty cut vector. I then transform JM101 with the
products of the ligation reactions. (I should mention that I EtOH ppt, wash
w/70% EtOH and resuspend the ligated DNA in water to cut down on salts
before electroporation.)

The puzzle comes when I pick several transformants from each time point and
analyze miniprep DNA for clones of the right size. Each time point always
reveals a radical range of differently sized clones with little
correspondence between the timepoint and the size of the deletion clones
observed. As a result I'm forced to pick, miniprep and analyze lots of
clones in order to get a series of deletions spanning my cDNA. I'm not
griping so much as I'm curious why there isn't a better correspondence
between timepoint and resultant size of deletions. My colleagues have told
me this result is typical. The poor Promega tech said he hadn't heard of
this happening and would check into it (he still hasn't come back to me.)
My best explanation is that the products of Exo III deletions at each
timepoint are a range of sizes based on the fact that not all of the DNA is
bound by Exo III at the same time. If the products at each time timepoint
were plotted as size of DNA vs frequency, I would expect to see a skewed
left distribution, as the size distribution would tail out to those
molecules that had been barely digested.

According to this thinking, it would seem that the concentration of Exo III
is limiting, and that I would probably get a smaller range of clones at
each timepoint if I increased the Exo III concentration.

Please comment on whether this result is typical; and, if not, how I can
rectify the situation. I'm tired of all these minipreps.

P.S. I've also found that I need to transform E. coli with the ligated DNA
immediately if I'm to get a decent number of transformants. The products of
the ligation reactions do not seem to store well, even as pellets under 70%
EtOH. Why is this?

-- 
Casey M. Finnerty
Boyce Thompson Institute at Cornell University
cmf5 at cornell.edu



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