13. Title : Genetic checks for the Gal strains used in the Biotek#

13.1. Date#

30092021-

13.2. Objective#

  • To check for the presence of the pgal promoter in front of CDC42 for the gal strains

  • To check for the abscense of the endogenous CDC42 promoter in the gal strains.

  • To check for the abscense of BEM1 and BEM3 in the mutants .

13.3. Method#

  • gDNA extraction of ywkd065 a/b and ywkd071I/II

  • PCR

  • Primers :

13.4. Results#

  • gDNA extraction

    • Using 3 eppies with 2mL of culture for pellet

    • Elution of 20ul per eppi

strain

concentration

ywkd065a

47.6ng/ul

ywkd065b

51ng/ul

ywkd071I

40ng/ul

ywkd071II

40ng/ul

  • There is are suspicious bands when checking the cdc42 promoter. The primer pCdc42 downstream does not bind to the pgal construct DNA, hence I do not get the presence of this band…

Checks for ylic139 and ylic140

  • It is streaking that the mutants ylic139 and ylic140 do have BEM1!!!! (aneuploidy???)

  • It seems ywkd071I does not have the endogenous CDC42 promoter. So it is good.

Important note

  • The primers olic60/61 were wrong!!! they were binding to kanmx cassette instead of BEM1. That explains the bands in the mutants..

  • I ordered new primers that bind to BEM1 upstream and downstream . Let see..

It shows that the mutants indeed have BEM1, which is cumbersome

13.4.1. PCR on control strains#

  • original strain used for transformation (yll117) does not show a band for BEM1. This is good.

  • The strain from Thomas ytw001: \(\Delta\)bem1\(\Delta\)bem3 seems to have both (I dont understand this), also my strains ylic141/ylic142 , \(\Delta\)bem1\(\Delta\)nrp1/\(\Delta\)bem1\(\Delta\)bem3\(\Delta\)nrp1

13.4.1.1. DNA extraction for ywkd017#

ywkd017:

  • \(\Delta\)bem1\(\Delta\)bem3\(\Delta\)nrp1

  • Positive control for all markers

  • gDNA concentration: 76ng/ul , 80ul Volume

13.5. Conclusion#

  • we may repeat the BEM1 transformation for :

    • ylic139

    • ylic140

    • ylic141

    • ylic142

    • ytw001