use of S1 nuclease to blunt-end DNA

Marc Crepeau mwcrepeau at ucdavis.edu
Thu Apr 23 17:41:59 EST 1998


   I am wondering if anyone can provide me with some tips on using S1
nuclease to remove 3' overhangs from DNA. I tried to do this recently
using the time-honored non-quantitative approach of 1ul enzyme in a 10ul
reaction with incubation at 37C for one hour. The result was that my DNA,
both single and double stranded portions, was completely digested.
   That was when I decided to learn a little more about the enzyme. The
first thing I found out is that heating is not recommended for this
application (even though the activity increases dramatically at elevated
temperatures) because any melted portions of the DNA will quickly be
digested. I also learned that even at low temperatures the specificity for
ssDNA is lost if high concentrations of enzyme are present. Recommended
enzyme concentrations seem to be about 0.1-0.2U/ul (versus the 40U/ul that
I used on my first try). Which leads to my question:
   What should I dilute this enzyme with? My Boehringer Mannheim enzyme
didn't even come with a reacton buffer, much less a dilution buffer. It is
stored in 20mM Tris, 50mM NaCl, 0.1mM ZnCl2, 50% glycerol, pH7.5. Do you
think it would be happy diluted about two-hundredfold in this buffer? I
ask because I know some enzymes aren't happy when stored diluted. I'd like
to be able to store the diluted enzyme. It would be pretty wasteful to
dilute down 400U (1ul) and then have to throw 398U away!


Marc Crepeau
<mwcrepeau at ucdavis.edu>



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