17. Title : Contruction dbem1dbem3+pGal from ylic139 -> ylic140#

17.1. Date#

30062021-15072021

17.2. Objective#

  • To get a clean dbem1dbem3+pgal:CDC42 because ywkd073I and II is growing miserably.

17.3. Method#

  • Transformation protocol on ylic139: ywkd071+bem1:KAnMX

  • Use ywt04gDNA with oll401/402 primers, bem3::NAT to get the PCR DNA for transformation.

    • 1ul ywt04 template

    • Leila_60 PCR protocol

    • 8 PCR to merge and have more DNA

  • make 10 SC-URA(4x)+ 2%Raff+2% Gal + G418+NAT as selection plates for the transformation.

  • Incubate 10ml ylic139 in 4x CSM+2% Raff +2% Gal + G418 (20ul 500X) (11:00)

  • PCR purification , 4 eppis , elution 20ul for each eppi. (I used 200ul for binding buffer since the product is bigger than 2000kb)

Note : I should have used primers olic51/52 to build the DNA for transformation because primers olic401/402 are more used to check the location of the insertion after transformation.

  • same PCR with olic51/52 for transformation

    • the gel did not show any band …. I dont get why…

    • repeat()

17.4. Results#

  • Expected size for the PCR (though the gel was not great)

  • Purified PCR concentration: 87 ng/ul , having around 75ul , which gives as a total amount approximately : 6.5ug

  • For Transformation I should around 2ug , so around 25ul .

  • Expected size for the PCR with olic51/52

  • Purified PCR concentration: 90 ng/ul , having around 40ul , which gives as a total amount approximately : 3.6ug

  • For Transformation I should around 2ug , so around 22ul .

17.4.1. Incubation results#

  • Not growth after overnight liquid incubation by ylic139.

Change of strategy: Plate ylic139 in CSM plates with Gal and G418 and take a single colony to inoculate in liquid culture. (01072021)

  • I used already made CSM+0.1%Gal that I added 1.5ml of 20% gal . Once they get dry I will add 30ul 500x G418 and streak the cells from glycerol stock.

    • There were cells on the plate after growth during weekend, so I took two single colonies, from ylic139a and ylic139b and inoculate in 4xCSM-NF+2% Raff+2% Gal+G418 500x(20ul) at 9:30.

17.4.2. Transformation#

  • OD measurement on 06072021

Time

Strain

OD- 2x diluted

real OD

11:00/06072021

ylic139_a

0.275

0.55

ylic139_b

0.137

0.27

15:00/06072021

ylic139_a

0.279

0.56

ylic139_b

0.222

0.44

08:45/07072021

ylic139_a

0.3

0.6

ylic139_b

1.127

2.24

  • Transformation with PCR (ywt04/olic51/52) on 07072021

    • 25ul of DNA from purified PCR -> 25ul*90ng/ul=2.25ug

    • Recovery step , from 10:30->12:30 in 4x CSM+2% Raff +2% Gal

    • Plating:

      • selection plates: SC-URA(4x)+ 2%Raff+2% Gal + G418+NAT

      • After the recovery I centrifuged the samples and resuspend them in 100ul MiliQ .

        • I plated the transformed cells 50ul from this tube , 0X dilution.

        • I added 950 ul MiliQ to the rest and plate 50ul in one plate and 200ul in other one.

        • Plate 200ul in CSM+0.1% Gal to check viability.

        • Plate 50ul of 0x non transformed cells in selection plates.

        • Plate 200ul of 10x non transformed cells in CSM+0.1% Gal to check for viability.

  • Transformation plates

  • Very few colonies , it seems it was a hard transformation.

  • Colony PCR

    • 8 colonies, colony 2 and 3 were big , the rest small.

    • Transfer colonies/ streak into CSM+0.1% Gal + NAT

    • 5ul MiliQ suspension, 10ul MiliQ for the big colonies

    • 1ul used for PCR (LEILA_60)

    • colony 3 seems the only right , which was a big colony. See the growth in CSM+0.1% Gal +NAT to store it in glycerol stock.

  • Re-streak and liquid incubation

    • restreak of the colonies for PCR in CSM+0.1% Gal +NAT

    • Inoculate colony 2 and colony 3 (the ones that grew and big colonies) in CSM+2% Gal+G418+NAT (5mL) for glycerol stocks.

17.4.3. PCR with gDNA#

  • DNA extraction of ylic140a (colony 2) and ylic140b (colony 3)

  • Inoculation in 10ml of 4X-NF-CSM+2% Gal+NAT+G418

  • Extraction of 4 eppies with pellet from 2ml of culture.

  • Elution of 20ul per eppi.

PCR

  • Check with oLL401/402 primers

  • Check with olic56/57

  • Include ylic139 as a negative control for the presence of BEM3::NAT (expected length around 4kB) and as a positive control for BEM1::KanMX (around 3kb)

  • 5ul loaded of gDNA

17.5. Conclusion#

  • The dna is ready for transformation

  • At least one colony is correct .

  • strain name : ylic140 MAT \(\alpha\) can1-100 leu2-3,112 his3-11,15 BUD4 from S288C bem1::KanMX bem3::NAT CDC42::URA3-GAL1pr-CDC42,CAN1::MFAprHIS

    • two biological replicates , a (colony 2 check again by PCR) and b (colony 3 checked by colony PCR)