81. Title : 17072019-DNA extraction of ylic133: ura3- ade2- clones anad PCR :ok: :white_check_mark:#

81.1. Date#

Wednesday 17072019

81.2. Objective#

To extract the DNA to precisely check that neither ade3 nor ura3 is in the genome.

81.3. Method#

  • DNA extraction KIT EurX

  • Elution of clones 1-3: 50ul because they were dense after overnight culturing.

  • Elution of clones 4-5:10ul because they were not dense at all after overnight culturing.

  • DNA nanodrop

    ylic133_1: 28.9 ng/ul

    ylic133_2: 32.8 ng/ul

    ylic133_3: 16.9 ng/ul

    ylic133_4: 2.7 ng/ul (This one I had a very small pellet at the starting)

    ylic133_5: 6.5 ng/ul (This one I had a very small pellet at the starting)

    ylic133_6: 4.6 ng/ul from 19072019 extraction(not good quality DNA, 260/280=2.42,260/230=1.10)

    ylic133_7: 7.7 ng/ul from 19072019 extraction(regular DNA quality,260/280=1.9,260/230=1.7)

    ylic133_9: 9.3 ng/ul from 19072019 extraction(not good quality DNA, 260/280=2.32,260/230=1.94)

    ylic133_6: 7.8 ng/ul from 22072019 extraction (not good quality DNA, 260/280=2.4,260/230=0.94)

    ylic133_7: 7.1 ng/ul from 22072019 extraction(not good quality DNA, 260/280=2.65,260/230=0.84)

    ylic133_9: 6.9 ng/ul from 22072019 extraction(not good quality DNA, 260/280=2.16,260/230=0.92)

  • PCR with primer 22 and primer 23 of all of them , using 1ul of templates, each of the DNAs.

81.4. Results :)#

  • DNA Gel 😁😁 The bands are ALL IN THE RIGHT LENGTH of colonies from 1-5

  • The 1st PCR using clones 6,7 and 9 DID NOT WORK , I DID NOT GET ANY BAND :( 😕

  • The 2nd PCR just work clone 7 and very faint band.. something was not right with the genomic prep this time.. :(

  • The 3rd PCR with the DNA extracted from 19072019 and from 22072019, using 1ul of template ine ach of them.

81.5. Conclusion#

  • These clones are not pink in the re-streaking plate in YPD after 2 days of incubation.. so if they dont have the right deletion I should take the other ones 6, 7 and 9 which are pink.

  • I will do also the genomic prep for the pink clones from the 5FOA plates, namely , clones, 6,7 and 9.

  • After the repeat of the genomic prep for clones 6, 7 and 9, we can see that the clones 7 and 9 are the correct ones, meaning that they are the ones that have the ura kickout and not an URA mutation as clone 6.