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
