# Title: 27082019-Sporulation protocol of 8 re-streakings from ylic133_5+yEk7a 😔

# Date

27082019-06092019

# Objective

To test the ability of mating using liquid drops of ylic133_5 and yEK7a

# Method

• Sporulation protocol
  • Incubation in YP+KAc @ 9:30am
  • Incubation in MiliQ+KAc @ 17:45, total of 8 tubes.
• Selection Protocol 02092019
  • 10x and 100x dilution of spores of the 8 tubes in YPD+G418+Hygro+NAT
  • 100x dilution in YPD as positive control
• I want to select for those haploids that grow in G418+Hygro+NAT and they are ade2 defficient. (pink colonies in YPD+G418+Hygro+NAT )

# Results

• The efficiency of sporulation was very low, less than 20% of the cells did sporulate, from the 10X microscope inspection.
• Still with this low number, I decided to proceed with th selection protocol.
• The selection protocol did not work, I did not found any colony growing even in the YPD plates after 4 days of incubation.

# Conclusion

• It seems I kill all cells during the selection protocol, because most of them were haploids and with the zymolyase treatment I kill them.
• There is a problem , still unsolved with the mating process between those strains.

# Next steps

• Repeat it using 2 more positive controls : yEK7a+yll3a (alpha type), and ylic133 + ylic128 (mating type a)