Random sporulation
1. Grow a 2 ml overnight culture of the diploid strain.
2. Wash 350 µl of this overnight culture once with 1 ml H2O.
3. Resuspend pellet in 3-5 ml sporulation medium and shake at 20-30C for 5 days in a glass tube (do NOT use plastic) and make sure that cells get oxygen. Some strains sporulate better than others; the temperature may have to be optimized for strains with poor sporulation efficiency, such as Winston S288C, or for mutants with TS alleles, but usually 30C works fine.
4. Spin down 1 ml of sporulated cells in an Eppendorf tube. Check whether cells have sporulated by light microscopy and document sporulation efficiency.
5. Resuspend in 50 µl spheroplast buffer, add 1-5 µl zymolase (10 mg/ml stock) and incubate at 30C for 30 min.
6. Vortex 2 min such that the liquid swirls nicely (do NOT vortex such that liquid moves violently through the tube). The hydrophobic spores will stick to the tube wall and form a ring (see illustration). This ring of spores is often not easily visible.
7. Wash 4 x 1 ml H2O by inverting the tube 5-6 times and make sure to remove as much H2O as possible between the washes.
8. Elute the spores by adding 500 µl NP40 0.01% and vortex briefly.
9. Plate 100 µl on a YPD plate or on a selection plate (in case of antibiotic selection such as G418, hygromycin or nourseothricin, the spores can also be plated directly onto the selection plate). The remaining spores are stored at 4C and usually stay viable for several weeks.
10. Incubate plate until colonies appear. Make sure that there are not too many colonies on the plate to prevent cross-contamination (50-100 colonies should be the maximum). If too many colonies appear, plate less spores.
11. Pick colonies and patch onto a fresh plate and genotype.
Buffers
Sporulation medium:
1 % KAc
50 µg/ml adenine
Filter sterilize
Sporulation medium can be stored at RT for at least 6 months without notable loss of sporulation efficiency.
Spheroplast buffer
50 mM KH2PO4 pH 7-7.5
1.2 M sorbitol
Filter sterilize
Buffer can be stored at RT for at least 6 months.
2. Wash 350 µl of this overnight culture once with 1 ml H2O.
3. Resuspend pellet in 3-5 ml sporulation medium and shake at 20-30C for 5 days in a glass tube (do NOT use plastic) and make sure that cells get oxygen. Some strains sporulate better than others; the temperature may have to be optimized for strains with poor sporulation efficiency, such as Winston S288C, or for mutants with TS alleles, but usually 30C works fine.
4. Spin down 1 ml of sporulated cells in an Eppendorf tube. Check whether cells have sporulated by light microscopy and document sporulation efficiency.
5. Resuspend in 50 µl spheroplast buffer, add 1-5 µl zymolase (10 mg/ml stock) and incubate at 30C for 30 min.
6. Vortex 2 min such that the liquid swirls nicely (do NOT vortex such that liquid moves violently through the tube). The hydrophobic spores will stick to the tube wall and form a ring (see illustration). This ring of spores is often not easily visible.
7. Wash 4 x 1 ml H2O by inverting the tube 5-6 times and make sure to remove as much H2O as possible between the washes.
8. Elute the spores by adding 500 µl NP40 0.01% and vortex briefly.
9. Plate 100 µl on a YPD plate or on a selection plate (in case of antibiotic selection such as G418, hygromycin or nourseothricin, the spores can also be plated directly onto the selection plate). The remaining spores are stored at 4C and usually stay viable for several weeks.
10. Incubate plate until colonies appear. Make sure that there are not too many colonies on the plate to prevent cross-contamination (50-100 colonies should be the maximum). If too many colonies appear, plate less spores.
11. Pick colonies and patch onto a fresh plate and genotype.
Buffers
Sporulation medium:
1 % KAc
50 µg/ml adenine
Filter sterilize
Sporulation medium can be stored at RT for at least 6 months without notable loss of sporulation efficiency.
Spheroplast buffer
50 mM KH2PO4 pH 7-7.5
1.2 M sorbitol
Filter sterilize
Buffer can be stored at RT for at least 6 months.