William_D_WARREN at UMAIL.UMD.EDU
Wed Jun 17 17:11:00 EST 1992
>We had a slight problem with ExoIII deletions and disappearing DNA as well.
>The problem in our case was that according to the procedure the ExonucleaseIII
>needed to be heat denatured at 70C before the S1 nuclease step and if you are
>not careful to let the DNA cool SLOWLY after the denaturation then the S1 will
>chew through any unannealed DNA. We repeated the procedure many times before
>realizing this problem. We weren't using the kit but you might pay close
>attention to how you're carrying out this step of the procedure.
The original querie stated that they were using the Erase-a-base
kit from Promega. The instructions that accompany the kit call for time
point samples to be taken and added to tubes containing S1 nuclease
and a low pH buffer (pH 4.6 - conditions under which ExoIII will not work).
There is no heat step in the protocol before the S1 digestion, only
after. I seem to remember someone in the lab forgot to add the
S1 stop buffer (high pH and EDTA - conditions that stop S1 digestion)
prior to the S1 heat inactivation one time and their DNA was degraded.
- Bill Warren
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