Need Info on Zymolyase for Tetrad Analysis

R. Bassett rbassett at blue.weeg.uiowa.edu
Mon Jan 15 11:45:35 EST 1996


In regard to your questions about tetrad dissection--there are many 
different protocols.  The one I have personally had the most success with 
uses a solution of 5 mg/ml zymolyase dissolved  in 1M sorbitol (you will 
have some debris remaining in the bottom of the tube, you can remove it 
or simply leave it).  As for buffers and times, a lot depends on whether 
you are sporulating in liquid media or on plates.  For liquid, we spin 
down about 200ul of sporulated culture, wash &  resuspend in 1M sorbitol, 
and add the zymolase, incubate at room temp, then put on ice.  The amount of 
enzyme and incubation time are extremely variable depending on the strain 
you are using, and it just takes some fiddling around to find the right 
combination.  It seems that 16minutes  has been the shortest, up to 22 
minutes  with 10-30ul enzyme.  If using plates, simply take a loopful  of 
sporulated culture and suspend in 50ul of enzyme (no buffer) for about 
the same amount of time as before.  The plate method seems to work much 
better than liquid, and you also avoid some contamination problems.  If 
you have any other questions, I'd be glad to help.

R. Bassett, U. of Iowa



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