mung bean nuclease

brett brett at BORCIM.WUSTL.EDU
Sun Dec 10 19:39:59 EST 1995


>------------------------------------------------------
>   a.k.a.
>
>Chris Hoyle
>   Department of Biochemistry and Molecular Biology
>University of Leeds             phone +44 0113 2333172
>Leeds LS2 9JT                   FAX   +44 0113 2333167
>Great Britain           e-mail bmbckh at biovax.leeds.ac.uk
>------------------------------------------------------
>I've been trying to remove single-stranded extensions from plasmid DNA
>after digestion with EcoRI without success. The mung bean nuclease was
>purchased from NEB. DNA was suspended at 100ng/ul in NEB buffer 2 in
>presence of 1/10th vol. of ZnSO4 (supplied). 1 unit of enzyme was added
>and mixture incubated at 30C for 30min. 20 transformants were screened
>for absence of EcoRI restriction site in isolated plasmid DNA. All
>isolated plasmid DNA was cut with EcoRI. Anyone got any aadvice,
>recommended protocols, etc.?

I was literally just setting up a MBN digestion and reviewed some previous
constructions I made with this enzyme. Once I was killing a NcoI site, and
found that I had to overdigest 20x to get good chewing back efficiency.
Furthermore, if the region around your site is A/T-rich, bring down the
temperature to 25oC, as I experienced excessive chewing at ends that were
breathing. I'm sure you'll
be able to find conditions for generating the ends you want.


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




More information about the Methods mailing list