2. Dilute into YPA medium (1% yeast extract, 2% Bacto-peptone, 2%KOAC) to OD600~ 0.2, Vigorous shaking.

3. For ts mutants, ~15hours at 23C, others ~10 hours at 30C, to reach OD 1.2-1.4 (un-budded cells ~85%. OD is important. Do not dilute back if overshoot. ).

4. Wash in pre-warmed H2O once, then transfer cells to same volume of SPM medium (2% KOAC). Pachytene cells will enrich after ~5 hours sporulation with wt.

5. For ts mutants and controls, pre-incubate at 23C for 1-2 hours, then shift up to 34C or other desired temperatures.

Reference:
Cha, et al., 2000, Genes & Dev. 14:493-503