How I get Mitos!!!

[Kathy Baynton]

Dear Lothar,

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) 

						Sorbitol 									1M
			   Na Citrate        0.1M
						EDTA              50mM
	     beta-mercapto     14mM

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!

Kath