Mung Bean Nuclease

brett brett at BORCIM.WUSTL.EDU
Tue Feb 6 12:13:14 EST 1996


>Hello!
>
>I seem to have big problems with Mung Bean Nuclease digestion. It looks as
>if it degrades my double stranded DNA - I just wanted it to chew up the
>overhang left by BamHI. I use very little of enzyme - 100 times less then
>recommended by Maniatis, but it still does not work. Anyone having
>experience with this enzyme?

Well, you don't provide many specifics about your conditions, but yes, I have
seen MBN shall we say, overacheive? However, never did I see it completely
degrade my DNA. Rather, it may take off an extra base or two during blunt ending
for cloning. Things seem to improve at room temperature, perhaps due to less
breathing at the ends. Even with a 10 fold overdigestion (by units), I was
able to obtain the ends I needed. However, I recommend watching the time of
incubation. Perhaps you could pull out aliquots over time, stop MBN with
some
EGTA, and checking your DNA quality. Try using the fragments at some
intermediate time, before degradation appears to be a problem. The other
thing 
to question is the quality of your DNA. Perhaps there is a contaminating
nuclease that likes Zn++, which would also be easy to check. If so, clean it up.
Good luck.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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