# Title : 12092019-Transformation of ylic133 with the pLL112 plasmid 😔

# Date

12092019-20092019

# Objective

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

# 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

# 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.
  • 

# 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... 😔
• 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.