96. Title: 06062019-Yeast transformation with the OEP1 :white_check_mark: :white_check_mark:#

96.1. Date#

06062019

96.2. Objective#

To remove the ade2 gene from yll3a and insert the URA+ promoter in its locus.

96.3. Method#

Yeast transformation protocol

96.3.1. Homology arms:#

  • left homology arm: 236 bp

    PCR of primer_1_new_upstream_forward and primer_2_upstream_reverse

  • right homology arm: 272 bp

    PCR of primer_5_downstream_forward and primer_6_new_downstream_reverse_NO_Rga1

96.3.2. DNA concentration#

8ul of 803 ng/ul OEP1 = 6.4 ug

96.3.3. Selection plates#

  • -URA + 3x ADE

  • -URA

  • YPD

96.4. Results#

Growth of pink colonies in -ura+3x ADE plates :-)

No Growth of pink colonies in -ura plates 🤔

96.4.1. Genomic Prep#

96.4.2. Expected size of the insert in PCR: 2065 bp#

96.4.3. Expected size if the insert is NOT in the ade2 location: 2739bp#

96.5. Results 😀😀😀#

96.6. Conclusion#

  • To see the adenine deletion phenotype, namely, see pink colonies upon adenine depravation, in a transformations is necessary to supplement the transformation plate with extra adenine aminoacid. I did 2x more adenine than what the normal drop-out has.

    The usual concentration in CSM is 10mg/l

    I prepare a 50mL stock of 10mg/ml (1000x concentrated), and add 40ul for 20ml -URA plates (2x), which sum to 3x (2x+1x) the normal adenine concentration in a drop out plate.