The best way to sporulate is using liquid media. Overnight culture in YE is added (100 ul x 2 strains) to 1 ml of EMM₂ (without NH₄Cl). Overnight shaking at 30℃ will give you enough spores to work on. Spores are also formed on SPA agar medium after 2 days of incubation at 30°C. Ascospore forming on SPA media are suspended in sterilized distilled water containing Zymolyase 20T (Seikagaku Corp.; final concentration, 1 mg/ml), then placed at 37°C for 5 min, and transferred onto a YE agar plate to dissect the spores. Spore dissection is generally performed using an MSM300 dissection microscope (Singer Instruments). S. japonicus generates eight spores in each ascus. Dissection should be done ASAP as the spore start to germinate on YE and tend to conjugate with neighboring cells even at 4℃.

1) 50ml YE(+Ade, Ura) cultured up to 3x10⁶/ml(early log phase) at 30°C.
2) 15min on ice.
3) 3 times wash by H₂O on ice.
4) After removal of supernatant, add 1M sorbitol 10ml with 50mM DTT and mix it.
5) Incubate 30°C, 15min.
6) Spin down cells by 3000rpm, 3min, 4°C and remove supernatant.
7) Wash by 1M sorbitol 5ml.
8) Spin down cells by 3000rpm, 3min, 4°C and remove supernatant.
9) Add 1M sorbitol 50µl, DNA(0.5µg) and mix it well.
10) Stay it on ice for 30min.
11) Transfer 40µl cell mix to the cuvette(2mm).
12) Electroporation(2.3kV, 200Ω, 25µF).
13) Add ice cold 1M sorbitol 1ml immediately.
14)Culture the cell mix for recover with YE media at 30°C for overnight.
    (When use the plasmid containing kanMX, culture the cell mix for recover with YE(+Ade, Ura) media at 30°C.)
15)Plate it on selective media.