69. Title: 27082019-Sporulation protocol of 8 re-streakings from ylic133_5+yEk7a :pensive:#

69.1. Date#

27082019-06092019

69.2. Objective#

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

69.3. 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 )

69.4. 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.

69.5. 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.

69.6. Next steps#

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