86. Title : 11072019-ylic132-1 and ylic132-2 transformation with the OEP2 :white_check_mark:#

86.1. Date#

11072019

86.2. Objective#

To kickout the ura3 gene from the ade2 locus

86.3. Method and procedure#

  • I inoculated 100ml of cells of both strains on Monday 08072019

  • On Wednesday morning (10072019) I diluted the saturated cultures 10^5x times, 1ul in 100ml YPD.

  • Next morning, 11072019, strangely, ylic132_1 was really dense with OD of 16 and ylic132_2 not dense at all, with OD=0.6 🤔

  • I diluted ylic132_1 , 80x to approximately OD of 0.2 and I didnt dilute ylic132_2, just add 2x ADE, which means 200ul of 1000x concentrated stock to a 100ml culture.

  • I had diluted again ylic132_2 50x because at 11:30 it was close to OD=1,2, so it seems the adenine speed up the growth. While ylic132_1 was just 0.252 , so I added 200ul 1000x ade to the culture.

  • at 13:15 the OD was 1.38 for ylic132_2 and 0.9 for ylic132_1.

  • after centrifugation I realized ylic132_1 was contaminated 😬 😩 because the supernatant was not completely clean as ylic132_2 and the pellet was kind of yellowish.

  • I used for all centrfugation steps 600g and 5 secs , and also I minimize the time in LiAc 0.1M.

  • I pipette in and out after adding every component of the transformation mix , NO VORTEX ☠️

86.3.1. Homology arms:#

  • left homology arm: 252 bp

    PCR of primer_1_new_upstream_forward and primer_7_upstream

  • right homology arm: 271 bp

    PCR of primer_6_new_downstream_reverse_NO_Rga1 and primer_8_downstream

86.3.2. DNA concentration#

I used 10ul of 336.8ng/ul OEP2 , so 3.36ug of DNA

86.3.3. Recovery Step - Essential for plating in selective 5FOA plates#

In order to use the two options for recovery, namely, previous 2-4 hours incubation in 1ml YPD and then plating in 5FOA plates , and plating 200ul in YPD plates and incubating the rest in 30C overnight for plating on the next morning , and the ones in the YPD plate do replicate plating in 5FOA on next morning. I did:

  • After removing the transformation mix , resuspend the cells in 1mL YPD

  • Split 500ul to a new eppi, to have two eppies per recovery options

  • Add 500ul YPD to each eppi

  • Take 200ul for one of them and plate in YPD+3xade (60ul ade 1000x)

  • The rest ,2 tubes for ylic132_2+OEP2 and 2 tubes for negative control of ylic132_2 , are left in the 30C incubator, from 3:15pm.

  • At 5:30pm I took one of each tube and plate in 5FOA (I only have 3 5FOA plates.. so I think I can only plate the transformed cells 1mL) I need exactly 3 plates : one for after incubation, one for replica plate , and the other one for plating the rest of the overnight culture.

  • Next morning, I replica plated the YPD plate with a cell loan onto a 5FOA +3xade plate.

  • Plated from the overnight recovery culture of ylic132_2+OEP2, 800ul onto a new 5FOA +3x ade plate, to increase the chances of getting the right transformants.

  • Plate 800ul of ylic132_2 negative control onto another 5FOA+3xade I found in the 4C fridge.

  • Wait until 15072019, Monday, for the formation of colonies 🙏

86.3.4. Selection plates#

  • 5FOA plates + 3x ADE , in which only cells lacking URA3 are capable of grow. (positive control for the transformed cells)

86.4. Results -> I see colonies in 5FOA 😁#

86.4.1. Overnight recovery in liquid culture#

86.4.2. Replica plate from overnight YPD plate to 5FOA#

86.4.3. 3 hours recovery (one colony)#

86.4.4. Negative control - after overnight culture in YPD#

86.5. Next steps#

  • Colony PCR

    • 12 colonies in total: 5 from the overnight recovery, 4 from replica plating , the one from 3h recovery and 2 from the negative control.

    • Dissolving in 20ul MiliQ and replated in YPD

    • Using primers 22 (Primer 9 ADE Forward Check) and primers 23 (Primer 10 Reverse Check)

  • DNA Gel to see check bands according URA presence or not. Length for not presence 1046bp and otherwise 2065bp. IT SEEMS I HAVE SOME RIGHT COLONIES 😁😁

86.6. Conclusion#

  • Definitely, the recovery step is essential to get transformed cells in 5FOA plates

  • Now I dont see , this egg like structure of the yellow cells concentrated in the center and pink cells around. I dropped the extra adenine at the edge of the plate, and not dropping in the center.

  • The most efficient one is the overnight recovery in liquid culture, in terms of amount of colonies.

87. Next steps:#

  • Plating 8 colonies in -ade and -ura to check that they dont grow.

  • Overnight grow in liquid culture 1-5 for genomic prep.

  • Genomic Prep of 5 biological replicates

  • PCR with primer 22 and primer 23

  • Gel to check backgrounds

  • Sequencing to see that the URA is not there.