erase-a-base

ww40 William_D_WARREN at UMAIL.UMD.EDU
Wed Jun 17 14:13:00 EST 1992


>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.

Hummm... I have used this procedure with great success and not encountered
such problems. I bought the S1 nuclease and Exo III from Promega then
made the rest of the solutions myself :). Actually I was lazy and used small
scale mini-prep (alkaline lysis) plasmid DNA without problems although if
I ran a sample of DNA from each time point there was a faint background
smear that I attributed to nicked molecules. I have heard that some people
have had trouble with the S1 nuclease step and have opted for mung-bean
nuclease instead. A well designed experiment, taking samples at each
step for gel analysis, should allow your friend to pinpoint the cause
of the problem.

Good Luck

Bill Warren
Ctr. Ag. Biotech.
University of Maryland




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