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

# Date

07062020-

[ ] 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.

Colony PCR with freshly grown cells from glycerol stocks.
{#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.

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.
[x] 08072020- Plate all biological replicates of ylic135 in -leu2+6xade plates and YPD.
[x] 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.
[x] Gel 110V 25 mins

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 (?)

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.

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