# Title: 06062019-Yeast transformation with the OEP1 ✅ ✅

# Date

06062019

# Objective

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

# Method

Yeast transformation protocol

# 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

# DNA concentration

8ul of 803 ng/ul OEP1 = 6.4 ug

# Selection plates

• -URA + 3x ADE
• -URA
• YPD

# Results

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

No Growth of pink colonies in -ura plates 🤔

# Genomic Prep

# Expected size of the insert in PCR: 2065 bp

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

# Results 😀😀😀

# 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.