Co-culture of Query Strain and Deletion Mutant strains.
1. Query / Reporter strains (Mat alpha) were grown in a 3 mL of YPD medium as overnight culture.
2. deletion /Test strains (Mat A) were grown as overnight culture in YPD with the antibiotic G418.
Mating the Query Strain with the DMA
5 µl each of WT reporter and individual deletion strain were inoculated into 400 µl of YPD in 96 deep well plate. The cells were co - cultured overnight at 30°C
MATa/α Diploid Selection and Sporulation:
4. MATa/α zygotes were inoculated in diploid selection medium with the antibiotic G418 in 96 deep well plates.
5. The diploid-selection plates were incubated at 30°C for 2 days. Under shaking conditions.
6. 10µl of cultured diploid cells was reinoculated to enrich 400 µl of sporulation medium in 96 well deep well plates.
7. Sporulation plates were incubated at 22°C for 5 days in static.
MATα Meiotic Progeny Selection:
8. Spores formed in sporulation medium were transferred to HSM (SD – leu/Arg/Lys/Ura along with canavanine, thialysine + G418) to select out the desired meotic progeny.
9. Haploid-selection plates were incubated at 30°C for 2 days.
10. MATα meiotic progeny were further incubated in HSM (SD – leu/Arg/Lys/Ura along with canavanine, thialysine, G418) for a second round of haploid selection and was incubated at 30oC for 1 day.
For Sporulation medium
2 g histidine,10 g leucine, 2 g lysine, and 2 g uracil were mixed thoroughly and used for preparing medium as follows:
Chemical compound Quantity
Potassium acetate (1%) 1 g
Yeast extract (0.1%) 0.1 g
Glucose (0.05%) 0.05 g
Amino acid supplement
powder (0.1%) 0.1 g
The above composition was used for 100ml sporulation medium.
For Diploid Selection Medium (DSM –with glutamic acid w/o lys,arg,leu,ura) synthetic
medium (mentioned earlier) with amino acid mix w/o lys-arg-leu-ura , leu,G418 were used.
Haploid Selection Medium (HSM) was made with synthetic medium (mentioned earlier)
along with amino acid mix w/o lys-arg-leu-ura ,leu , G418, canavanine (50 mg/l), thialysine (50mg/ml). use clonat as well if your strain has nat-resistance marker.
In each case the medium was autoclaved before use.