michael coyne michael.coyne at
Tue Jun 16 22:38:15 EST 1992


I have again been asked by a colleauge without access to this net to see
if an answer to a problem can be found.  Any help or hints would be
greatly appreciated.

She has been using Promega's Erase-a-Base kit to chew into a large
insert she has, to make it available to sequencing.  As I understand it,
this kit allows one to create a series of nested deletions, such that
sequencing of a large fragment can be performed, with the overlaps
serving to align the various deletions created with the kit (I don't do
sequencing myself, so if this is inaccurate, the fault is mine). Anyway,
the protocol involves linerizing the clone with enzymes such that one
end of the insert is subject to digestion by Exo III, and the other is
protected by a 4-base 3-prime overhang.  After the vector is thus
linearized (and the insert has the proper ends), Exo III is added, and
aliquots are removed at timed intervals, and transfered to tubes
containing S1 DNA polymerase, where the recessed ends are filled.  The
thus shortened inserts are then re-ligated into vector, re-introduced
into the receipiant strain, and sequencing proceeds.

Her problem:  At the end of the Exo III digestion, her DNA is gone.  She
runs a test agarose gel of a small aliquot, and there is no DNA there.
She starts with plenty of CsCl-purified plasmid DNA, follows her
restriction digestion with a phenol:chloroform extraction and ethanol
precipitation (samples taken at this point confirm the DNA is still
present).  The exo III digestion is done at 15C, and for not enough time
to account for digestion of the whole vector and insert.

We have been trying to trouble-shoot this without success.  Any ideas
why she is unable to retain the DNA?  Anyone else encounter problems
with this kit?

We thought that it might involve the plasmids being nicked
(single-stranded) and therefore vunerable to exo III at several areas,
but this does not seem to be the case.

Any suggestions would be appreciated (except, please no attacks about
using a kit....!)


michael.coyne at

 ~ SLMR 2.1a ~ Nothing is so smiple that it can't get screwed up.
Channel 1 (R)   Cambridge, MA

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