Generating Yeast Knockout/Epitope-tagged Strains

PCR of knockout cassette from plasmid

1. Set up a 50 µL PCR reaction using Phusion DNA polymerase as follows:

      5x Phusion Buffer              10 µL

      10 mM dNTPs              1 µL

      20 µM forward primer         1.25 µL

      20 µM reverse primer         1.25 µL

      plasmid DNA (miniprep)           0.5 µL

      Phusion DNA polymerase         0.5 µL

      Water                          35.5 µL

2. Run PCR reaction using the “Phusion” protocol on the BioRad PCR machine:

Initial Denaturation:

      98ºC for 30 sec

Repeat for 30 cycles:

      98ºC for 10 sec

      55ºC for 30 sec

      72ºC for 2 min

Final extension:

      72ºC for 8 min

Cool down:

      4ºC for 10 min

3. Check 3 µL of PCR reaction on a 1% agarose gel. 

Yeast Transformation

**Protocol is written for 20mL of culture, which is sufficient for 2 transformations.  Increase volumes appropriately for additional transformations.

1. Grow yeast strain overnight in 3mL of YPD at 30ºC.

2. Measure the OD₆₀₀ of the culture.  Dilute back to OD₆₀₀ ~ 0.2 in 20mL of YPD.  Continue growing at 30ºC until OD₆₀₀ ~ 0.4 to 0.8 (approximately 3-5 hours).

3. Pellet yeast cells in 50mL conical tubes at 3500rpm, 3 min.  Pour off supernatant.

4. Resuspend cells in 1mL of 0.1M LiOAc.  Transfer 500 µL to two eppendorf tubes. 

5. Pellet cells at 8000rpm for 2 minutes.  Aspirate supernatant.

6.  Add the following mixture to each tube:

      240  µL 40% PEG-3350

      30 µL 1M LiOAc

      10 µL ssDNA (boiled for 3 min, then kept on ice)

      5 µL water

      15 µL PCR product

Resuspend the cells in the mixture well by pipetting.  

7. Incubate at 42°C for 40 minutes. 

8. Pellet cells by spinning in minifuge for 30sec-1min. Aspirate supernatant.

9. Resuspend cells in 200 µL sterile water.  Plate on appropriate media. 

For KAN (G418) and NAT (Nourseothricin) selection:

Plate cells on YPD.  Let grow overnight at 30°C. The next day, replica plate to either YPD+G418 plates or YPD+NAT plates.  Let grow at 30°C for 2-3 days. 

For auxotrophic (-URA,-LEU,-HIS,-TRP) selection:

Plate cells directly on appropriate synthetic dropout media. Let grow at 30°C for 2-3 days. 

Solutions:

1M LioAc, sterile

40% PEG-3350, sterile **Make fresh every 3 months to keep transformation efficiency high

Colony PCR to check integration of cassette

The number of colonies to screen by colony PCR is variable, depending on the efficiency of the integration.  For use in 8-strip PCR tubes, include one wildtype control (negative) and screen 7 colonies.  If efficiency of the correct integration is low, increase to 15 colonies (2 8-strip PCR tubes).

1. Pick a single colony using a toothpick. Smear a small amount of yeast from the toopthpick in to the bottom of the PCR tube.  Streak the remaining yeast on the toothpick on to the appropriate selective plates. 

2. Once all the tubes have yeast in them, microwave the tubes on high for 2 minutes.  Immediately place them in the freezer for 30 minutes. (A minimum of 30 minutes in the freezer is required, but the cells can be stored in the freezer for 2-3 days if necessary.)  

3. Set up a master mix for 50 µL reactions as follows:

                                           x8.5 (8rxns)

10x Taq Buffer (NEB)                 5µL      42.5 µL

      10 mM dNTPs                    1 µL           8.5 µL

      20 µM forward primer               1.25 µL 10.6 µL

      20 µM reverse primer (oEG009)           1.25 µL 10.6 µL

Taq DNA polymerase                 0.25 µL 2.13 µL

      Water                                42.25 µL    359 µL

4. Remove yeast from the freezer and quickly resuspend each colony in 50 µL of reaction master mix. 

5. Run PCR reaction using the “Colony PCR” protocol on the BioRad PCR machine:

Initial Denaturation:

      95ºC for 3 min

Repeat for 30 cycles:

      95ºC for 30 sec

      55ºC for 1 min

      68ºC for 2 min

Final extension:

      68ºC for 5 min

Cool down:

      4ºC for 10 min

6. Check PCR by running 10 µL of each reaction on a 1% agarose gel.