TITLE: Transforming Yeast (Instructor Protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for 
 

The yeast transformation is adapted from Geitz and Schiestl:
 


[GUIDELINES]
We use dropout media components from Sunrise Science, but I'm sure media from other vendors is fine. Double-check, though, that your "yeast nitrogen base" doesn't contain amino acids and other things.

[STEPS]
SECTION: Prepare 250 ml of 2xYPD media
1. Put  250 mL of deionized water in a 250 ml or 500 ml bottle, then add:
10 g  
5 g  


SECTION: Prepare 250 ml of 2xYPD media
3. Autoclave at 121 °C for 30 min on a liquid cycle.

SECTION: Prepare 250 ml of 2xYPD media
4. Cool, then using good sterile technique, add 25 mLof a 40 Mass / % volume solution of   .
 


SECTION: Prepare frozen competent cell (FCC) solution
5. To a 15 ml conical centrifuge tube, add:
0.5 mL  
1 mL 
Deionized water to a total volume of 10 mL


SECTION: Prepare 250 ml of 2xYPD media
2. Cap the bottle tightly and shake to resuspend the powdered components.
 

SECTION: Prepare frozen competent cell (FCC) solution
6. Filter-sterilize the solution into a new 10 ml tube.

 

SECTION: Prepare competent yeast cells
7. The day before, transfer 5 ml of 2xYPD media into two round-bottomed test tubes. Pick a colony of yeast off of the plate into each test-tube and grow, overnight, on the roller drum or orbital shaker at 30 °C.  This is your starter culture.

 


SECTION: Prepare competent yeast cells
8. The morning of the prep, measure the OD600 of the starter cultures. Pour 250 mL of the 2xYPD media into a 500ml baffled flask. Add enough starter culture so that the final OD600 is 0.5.

SECTION: Prepare competent yeast cells
9. Shake   until the OD600 reaches 2.0.

 


SECTION: Prepare competent yeast cells
10. Harvest the cells by centrifugation 2900 x g, 5 min at room temperature in a swinging bucket (or fixed-angle highspeed) centrifuge.

SECTION: Prepare competent yeast cells
11. Decant the media from the centrifuge tubes back into the baffled flask. Resuspend the pellet(s) in 125 mL of sterile water (vortexing is okay). Then, centrifuge 2900 x g, 5 min .

SECTION: Prepare competent yeast cells
12. Decant the water back into the baffled flask. Resuspend the pellet(s) in a total of  l of sterile water. Again, centrifuge 2900 x g, 5 min 

SECTION: Prepare competent yeast cells
13. Aspirate or decant the water. Resuspend the pellet in FCC for a total volume of 2.5 mL

 


SECTION: Prepare competent yeast cells
14. Transfer 50 µL aliquots to sterile microcentrifuge tubes. Load into Mr Frosty's (or the styrofoam container) and freeze overnight at -80 °C 

 


SECTION: Prepare competent yeast cells
15. The next morning, transfer to a freezer box and store at -80 °C

SECTION: Miscellaneous preparation
18. Prepare the salmon sperm:
If necessary, dissolve the salmon sperm in sterile TE buffer at a concentration of 2 mg/ml. (This is best done in the cold room on a magnet stir plate.)
Make 55 µL aliquots in PCR tubes. (Strip tubes and a repeat pipettor make this easy.)
Denature by setting a thermocycler to hold at 95 °C and putting the PCR tubes in for 5 min, then moving immediately into an ice/water bath.
Store at -20 °C



SECTION: Miscellaneous preparation
19. Prepare the   solution:
Put 2 mL of deionized water in a 15 ml conical microcentrifuge tube
Add 5 g   
Add more deinized water until the total apparent volume is 10 mL
Shake vigorously to suspend the PEG evenly
Put on a roller (or nutator or rocker) until the PEG dissolves completely.
Add more deionized water to a final volume of 10 mL.





SECTION: Instructor Tips &amp; Common Student Errors
20. Instructor Tips
Particularly after transforming E. coli, students usually find this lab to be pretty straightforward.
Students still struggle with the unit analysis required to "compute the volume containing XXXX amount of DNA".  I usually provide a basic example, along the lines of "if I have a solution that is 5 g/ml and I need 10 g total, how much solution do I need?" and then suggest they use the same reasoning. I also offer to double-check their answers if they'd like.
A common issue is "not enough PCR", especially if there have been a failed transformation or two. (There's usually plenty of plasmid.) If there's more than 100 ng of PCR DNA, I usually tell students to go ahead and try transforming anyway, with the warning that things may not be efficient and they might consider retrying their PCR while the incubation is going.
Wait three full days before trying to pick colonies. Yeast double every 2-3 hours on dropout media like this, so it will take time before colonies are visible. They're usually visible (but small) after 2 days -- too small to do anything with.
When interpreting their results, I usually ask students to think about why we plated on both the leucine and uracil dropout plates. What does it mean when cells are growing on one or the other? (FYI: the leucine plate is the transformation control -- it tells us whether we were successful at getting the DNA into the cells. The colonies on the uracil plate, though, necessitated both successful transformation and successful genome editing -- they're the colonies we want to move forwards with.)
SECTION: Prepare dropout media
16. Prepare 500 ml of synthetic defined media without leucine:
0.855 g   
0.335 g   
2.5 g   
5 mL of 100X uracil solution 2 mg/mL   
10 g  

Make up to 475 ml with deionized water and autoclave 121 °C 30 min on a liquid cycle.

Cool to 60 °C, then add 25 mL 40 Mass / % volume   and pour or pipette into petri dishes.


SECTION: Prepare dropout media
17. Prepare 500 ml of synthetic defined media without uracil:
0.855 g   
0.335 g   
2.5 g   
5 mL of 100X leucine solution 12 mg/mL  
10 g  

Make up to 475 ml with deionized water and autoclave 121 °C 30 min on a liquid cycle.

Cool to 60 °C, then add 25 mL 40 Mass / % volume   and pour or pipette into petri dishes.

SECTION: Instructor Tips &amp; Common Student Errors
21. Common student errors
Didn't immediately thaw the cells in the hot water bath and remove the freezing medium. Yeast likes to freeze slowly and thaw quickly!
Students still struggle with the unit analysis required to "compute the volume containing XXXX amount of DNA" (see above).
Not enough plasmid or PCR (see above)