mung bean nuclease

Dr. Arthur L. Kruckeberg bmbckh at biovax.leeds.ac.uk
Sun Dec 10 09:37:40 EST 1995


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   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
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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.?



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