39. Title : Preparation for PCR check of BEM2 presence and absence in strain ylic135#

39.1. Date#

07062020-

39.2. Objective#

  • Repeat the test done on beginning of May to see if I get something usable…

  • Check the presence of the leu2 marker in the position of bem2 gene , and checking the bem2 absence in the mutants strains.

39.3. Method#

  • Colony PCR with freshly grown cells from glycerol stocks.
    Sketch of what is expected in the colony PCR{#fig:sketch-pcr}

  • 06072020

    • 10:00am Plate all glycerol stocks of ylic135 in -leu2 plates , and in YPD

  • 07062020

    • No growth yet in -leu2 plates .

    • Pink colonies in YPD

  • 08072020

    • Colony PCR at 10:30am - 12:30pm.

    • Protocol “Leila”

    • 5ul Template

    • Primers 49 y 50

    • I added 1ul of DNA template from yll3a as a positive control for Bem2 presence, with primers 47 y 48.

39.4. Important Note!!!!!!!!#

  • THESE PCR WOULD NEVER WORKS BECAUSE THE PRIMER SET TAKEN FOR EACH TEST WAS WRONG!!!!!!!!!!!!

  • I was taking only the pink or the blues primer set, looking at the picture above, hence I would never get a band and the region tested was completely wrong!!!

  • The right primer set combination is:

    • 41/49 and 50/42 primer set to test the presence of leu2 marker in the mutants. So 2 PCR per strain.

    • 41/47 and 48/42 primer set to test the presence of BEM2 in the WT.

  • For 5 biological replicates there are 10 PCRs to test the leu2::bem2 construct plus 2 PCRs for yll3a positive control. So 12 PCRs per test.

  • 08072020- Plate all biological replicates of ylic135 in -leu2+6xade plates and YPD.

  • 10072020- PCRs with the right primer set at 10:30am

    • Total : 24 PCRs

    • 12 positive controls for leu2:bem2 locus and BEM2 in WT

      • primer set 41/49 , 42/50 for the mutants and 41/47 and 42/48 for WT

    • 12 negative controls for not having BEM2 in leu2 locus and not having leu2 marker in BEM2 locus in WT.

      • primer set 41/47 and 42/48 for the mutants and 41/49 and 42/50 for the WT , yll3a genomic DNA.

  • Gel 110V 25 mins

39.5. Results#

  • Finally got some bands due to the use of the right primers!!

  • It seems the right strains are the slowest ones , colony 13 and 16. All of the rest , it seems they also have BEM2 there (weird..) and the leu2 marker in the right position.

  • It seems colony 8, 12 and 7 are diploids (?)

39.6. Conclusion#

  • colony 13 and 16 seems to be the right one that has leu2::bem2 and no BEM2 present.

  • They are also the slowest one from the Biotek measurement of population growth rates.

39.7. Next Steps#

  • Transform them with the plasmid pBK549 to do SATAY on them, hopefully also send the data as Enzo for sequencing.