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.
Ctr. Ag. Biotech.
University of Maryland
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