How I get Mitos!!!
baynton at esbs.u-strasbg.fr
Sat Jan 11 08:53:53 EST 1997
I use zymolyase on a regular basis to isolate plasmid DNA for filter
blots and I have found that cells are best digested in the
mid-logarithmic phase - the closer to and longer in stationary phase,
the harder they are to digest. Also, I've found that if the cells
aren't washed well with either dH2O or sorbitol prior to digestion,
enzyme activity is greatly inhibited, in particular by components found
in growth media.
Other conditions - we carry out digests at 37°C for at least 30minutes
(progress can be easily monitored under phase contrast microscope) and
the final concentration is 1-2mg/mL (1mg/mL is usually sufficient). Our
zymolyaze buffer: (final concentrations)
Na Citrate 0.1M
All but the beta-mercapto can be prepared ahead as a 10 times stock - we
add the beta mercapto just before use.
Hope something here helps you out!
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