10. Title : Genetic checks for a single colony from the stock of bem1:KanMx mutants#

10.1. Date#

29102021-

10.2. Objective#

  • To figure it out if the stok completely is unstable , that is , all the cells are aneuploidy after bem1:kanmx transformation.

  • If not , then store a new clean glycerol stock.

10.3. Method#

  • Plate from glycerol stocks in CSM+2%Raff+2%Gal+G418

  • Plates were tested with negative(yll3a) and positive control (ywkd017)

  • Take one single colony and inoculate in same liquid media over the weekend

  • gDNA extraction (ylic139b,ylic140a,ylic140b,yTW001a)

    • 2 eppies per strain

    • 30ul elution

    • 42-47 ng/uL DNA concentrations

  • PCR inside BEM1 and inside KanMx

    • olic56/61, olic57/60, oll29/olic57

    • 1ul DNA

  • glycerol stocks if OK

    • Only for yTW001a

Day 2:

  • Inoculate a 2nd colony from ylic139a(discarded)

  • Analyze ylic141a,b and ylic142a,b

10.4. Results#

The pgal mutants are unstable clones, yet, yTW001a seems to be rescued.

Bands downstream BEM1 but not upstream (???)

10.4.1. Sequencing#

  • PCR to prepare samples for sequencing:

  • yTW001b seems that do not have BEM1

  • I sent samples from ylic141a and ylic142a with olic60 and olic57 primers to sequencing.

  • I sent all the samples from PCR from olic56 and olic57 to sequencing.

Overlap with the downstream part of BEM1

Overlap with the PTEF promoter for the same strain

  • This is indicating that both strains ylic141 and ylic142 are aneuploidies, because they show the presence of PTEF from KanMx and the downstream region of BEM1 in the same strain.

10.5. Conclusion#

  • The pgal mutants are unstable clones, yet, yTW001a seems to be rescued.

  • To confirm that BEM1 is not in the SATAy mutants , I will sequence using olic60/olic57 and olic56/olic57 primers.

  • yWT001b seems that do not have BEM1.

  • ylic141 and ylic142 are aneuploidies.

  • Repeat the transformations fro ylic141 and ylic142 ,and check for every colony the presence of Kanmx and the abscence of BEM1.