TITLE: Yeast transformation
AUTHORS: is Sparrow

[DESCRIPTION]
Protocol was obtained from Ulschan Bathe and Kristen Van Geller from UF on May 2023 and optimized by myself.

This protocol is a variation on the highly efficient LiAc/SS/PEG method from Gietz et al., 2007 DOI: 10.1038/nprot.2007.13

[STEPS]
SECTION: Prep
1. 2 days before yeast transformation, start 3mL pre-culture of your yeast strain in appropriate selective media. Growth in SC is fine for this step.
SECTION: Prep
2. If the DNA to be transformed is for integration, linearize your plasmid the night before and store in the fridge or freezer the morning of the transformation. If you do, heat inactivate the enzyme.

Note: for integrations, a high amount of DNA is needed for efficient transformation. Make the following digestion:
55.5 uL plasmid (aim for a concentration of 90-300 ng/uL, more is better)
6.5 uL CutSmart/r3.1/NEB buffer
3 uL Restriction enzyme (BE SURE TO DOUBLE CHECK WHICH ENZYME YOU ARE USING)

Double check the expected cut patterns on a gel by running 1 uL of digest.
SECTION: Growth of yeast to exponential phase
3. Dilute pre-culture, 1:35 into 30 mL YPD per 3 transformations (875uL of pre-culture into 30mL YPD). Grow until OD600 of 0.6 (it is acceptable between 0.6 and 1.5) is reach (approximately 360 min)

Note: For slow growing strains, start the cultures the night before. For example, JH45 takes 9-12 hours to reach OD 0.6 from a starter dilution of 0.13.

Note: in my experience, addition of antibiotic is necessary at this step especially for slow growing yeast strains as this helps prevent contamination.

Note: when transforming plasmids, the OD matters less and as long as it is somewhere between 0.4 and 1.5 you will get transformants, but when you are looking for integration, the ideal range is between 0.6 and 0.8.

Note: Yeast should have at least 2 divisions to maximize transformation efficiency, but not more than 4. Do not start a very dilute starter culture the night before unless you know that the yeast genotype is slow to grow and will reach desired OD overnight from 1:35~ dilution.
SECTION: Growth of yeast to exponential phase
4. When yeast reaches OD600 of 0.6, pour into 50 mL falcon tube and spin at 1500 x g, 5 min, 4 °C

During this step, put your ssDNA in a 95 °C water bath/heat block for 5 minutes, and immediately plunge in ice. Mark on the vial how many times the ssDNA has been boiled. Discard after 3 boils.

Note: pellet should be whitish, or off-white. A spot of brown at the center of the pellet is expected, but yellowish or brownish color overall is indicative of contamination.

SECTION: Growth of yeast to exponential phase
5. Decant supernatant
SECTION: Growth of yeast to exponential phase
6. Resuspend in 30 mL sterile milliQ water

SECTION: Growth of yeast to exponential phase
7. Spin at 1500 x g, 5 min, 4 °C
SECTION: Growth of yeast to exponential phase
8. Resuspend in 900 µL of TE/LiAc/water solution (make 1.5 mL: 150 µL 10x TE, 150 µL 10x LiAc and 1200 µL sterile water) and transfer to 1.5 mL tube. See materials for recipes.

SECTION: Growth of yeast to exponential phase
9. Spin at max speed 1500 0, 15 s, 4 °C
SECTION: Growth of yeast to exponential phase
10. resuspend in 300 µL TE/LiAc/water solution

Note: a 300 µL suspension will actually yield more than 300, so you may instead choose to resuspend in less volume (~233 µL ) to yield a total volume of 300 µL , which serves to concentrate the cells more.

SECTION: Addition of DNA
11. Aliquot 100 µLper transformation to individual tubes

Note: because of the volume of the cells, resuspension in 300 uL from previous step will yield more than actually 300 uL. 100 uL of this, even though not truly 1/3rd of resuspension is OK to use, or more can be used, but adjust the additives amount accordingly.
SECTION: Addition of DNA
12. Add, in the following order:
SECTION: Addition of DNA
12.1. 20 µL of 5 mg/mL salmon sperm ssDNA


Note: high quality ssDNA is critical - don't overuse your ssDNA
SECTION: Addition of DNA
12.2. DNA

For linear DNA: add 65 uL of overnight, heat-inactivated digest. You want upwards of 5 ug, and I have had success with 12 ug, but I do not know if there is an upper cap.
For plasmid DNA: around 1 ug of plasmid is sufficient.

If you are cotransforming with linear dsDNA (e.g., as a repair fragment for Cas9) generated from PCR, there is no need to PCR cleanup the fragment. Add the entirety of the PCR reaction alongside the plasmid DNA.
SECTION: Addition of DNA
12.3. Mix gently by flicking the tube
SECTION: Addition of DNA
12.4. 600 µL of "PEG mix" (see materials for recipe)

The PEG mixture must be made the day of with freshly sterilized PEG (do not autoclave, filter sterilize it)
SECTION: Addition of DNA
12.5. Mix gently by pipetting until homogeneous
SECTION: Incubation and outgrowth
13. Incubate at 30 °C for 45 min with rotation 
This can be allowed up to 3 hrs if necessary
SECTION: Incubation and outgrowth
14. Incubate at 42 °C for 20 min
This must be stuck to as close as possible 
SECTION: Incubation and outgrowth
15. Centrifuge at 700 x g, 5 min

SECTION: Incubation and outgrowth
16. Resuspend in 1 mL sterile water
Can also resuspend in less if low efficiency expected

Note : be certain of the sterility of your water!!! I suggest autoclaving fresh MilliQ water the morning of the transformation and stick it in the cold room 
SECTION: Incubation and outgrowth
17. Plate unto appropriate selection plates
 

Ensure that your plating method will keep everything sterile: use L spreaders or sterile glass beads.

Note: do not plate the entire volume of cells unto a plate. High plating density decreases growth and may also obscure selection if using minimal plates. Instead, I recommend pouring 3 plates for each transformation and plating 3:2:1 and saving some resuspension in the cold room just in case. This is especially important when selecting with auxotrophy, in which case 1/10-20th of the total resuspension buffer is typically enough to yield some transformants. You will not see any transformants if you plate all your cells in one plate, even if it worked.
SECTION: Incubation and outgrowth
18.