68. Title : 12092019-Transformation of ylic133 with the pLL112 plasmid :pensive:#

68.1. Date#

12092019-20092019

68.2. Objective#

To mark temporarily ylic133 in order to be able to select for diploids after the mating with yEK7a.

68.3. Method#

  • Plasmid transformation using pLL112: pRS416 (URA3 CEN6_ARSH4)

  • 12092019: Incubation of ylic133_2 in 10mL YP+2% Dextrose

  • 13092019: OD measurements 9:30am

    OD-10X dilution

    Titer

    Dilution factor to OD=0.5

    Time

    ylic133_2

    0.259

    2.6

    5.2

    9:30

  • 2h after the 5x dilution of the dense culture in 10mL YPD

  		•

    OD-10X dilution

    Titer

    Time

    ylic133_2

    0.126

    1.3

    11:30

    OD-10X dilution

    Titer

    Time

    ylic133_2

    0.239

    2.4

    13:30

  • 5ul of pLL112 plasmid , which has 2290ng/mL

68.4. Results#

  • 17092019- I found bacterial contamination on the plates :(

  • 17092019- The transformants on the -ura plate are extremely tiny colonies

  • 17092019 - Incubate ylic133-3 to redo transformation

  • 18092019 - The liquid culture was not dense enough to do the experiment.

  • 19092019 - I diluted the culture 1000x to do transformation next day.

  • 20092019 - Transformation with pLL112 (from a miniprep Ramon did the day before)

    • pLL112-148ng/ul , I used 10ul for transformation , hence I used 1.5 ug of plasmid

    • I plated two positive controls in -ura and all the negative control in -ura.

68.5. Conclusion#

  • I got very small transformed strains which are going to be named: ylic134=ylic133_3+pLL112 (mating type alpha, ura3+)

  • I inoculated transformants colonies into a CSM-URA liquid media and they did not grow , from an overnight culture. :(

  • It seems somehow they loose the plasmid… :pensive:

  • As next step I will transform ylic133 and yll3a as a positive control with pLL112, pLL55 and pLL56, to assess whether is the plasmid or the strain the bottleneck here.