mung bean nuclease

[brett]

>------------------------------------------------------
>   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.?

Oh yeah, don't just try to heat kill MBN, phenol extraction gave me much better
ligation efficiency (although, perhaps it was the Zn which interferred).


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