In article <1995Dec10.143740.22116 at leeds.ac.uk>, bmbckh at biovax.leeds.ac.uk
(Dr. Arthur L. Kruckeberg) wrote:
> 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 have had the same problem with NEB enzyme. I tried some dilutions,
nothing worked. I called up the company and got a replacement that didn't
work either. Finally, I bought enzyme from Stratagene, it worked like a
charm (although, way more expensive). I hate to suggest S1 instead, but
you may consider this option.