1. Tian He 2008. 07. 06

2. Media non-selective: YPD without antibiotics Selective: Synthetic complete dropout medium (SC) (auxotroph) YPD with antibiotics

3. YPD: 1000 mL Yeast Extract 10 g Peptone (Tryptone) 20 g Glucose 20 g Agar (for plates) 20 g Distilled H 2 O 1000 mL

4. Synthetic complete dropout medium (SC) 1000 mL Yeast nitrogen base without amino acids (YNB) 6.7 g Dropout mix (-His/-Trp/-Leu DO mix) 0.62 g Amino acids Glucose 2 g Agar (for plates) 2 g Distilled H 2 O 1000 mL

5. Cultivation Temperature: 30 ℃ Lower: yeasts grow slowly. Higher: yeasts die. Shake: 200 rpm or higher Avoid the contamination of E.Coli. Gloves are needed to avoid infection!

6. Notes: It takes 2 hours or more to complete a cycle. Yeast will age. Inoculate a new plate every month. The selectable marker and medium should be complementary!

7. Commonly used selectable marker LEU2, URA3, TRP1, HIS3, ADE1,2 pGREG 503 504 505 506 HIS TRP LEU ADE Question: why we use pGREG503 for homologous recombination? We cannot determine from the phenotype whether recombination occurs.

8. pGREG 503/4/5 Autonomously replicating sequence Auxotroph marker Antibiotic marker Homologous sequence Stuffer Promoter Tag

9. Yeast strain: AH109 GAL 4 induced promoters: GAL1, GAL2, MEL1

10. Transformation Plasmid/ssDNA/dsDNA The competent cells of yeast cannot be stored; it must be prepared before use!

11. Materials TE: 0.1M Tris-HCl, 0.01 M EDTA, pH 7.5 LiAc: 1M LiAc, pH 7.5 PEG: MW 4000 (50 % w/v), stored at room temperature. Capped securely to avoid evaporation. Single-stranded Carrier DNA(2.0 mg/mL): Salmon sperm DNA. Boil 1.0 mL carrier DNA for 5 minutes and quickly chill on ice water. Do not boil the carrier DNA every time. Keep a small aliquot in freezer box and boil after 3-4 freeze thaws. Keep on ice when out.

12. Protocol 1.Inoculate cells into 50 mL YPD and grow overnight to a density of 1- 2×10 7 /mL(nearly saturated). A suspension containing 1×10 6 cells/mL gives an OD 600 of 0.1. 2.Dilute to 2×10 6 /mL in fresh YPD and re-grow into exponential phase (1×10 7 /mL). It typically takes 3~4 hours. It is important to allow the cells to complete at least 2 divisions. Transformation efficiency remains constant for 3~4 cycles. 3.Harvest the culture in a sterile centrifuge tube at 2500 rpm for 5 minutes. Wash in sterile water twice. 4.Resuspend in 1.0 mL sterile water and transfer to 1.5 mL microfuge tube. centrifuge for 30 s at 13.000 g and discard the supernatant.

13. Protocol 5.Wash cells in 1.0 mL of TE/LiAc(10×) and resuspend at 2×10 9 cells/mL in TE/LiAc (1×) 6.Mix 50 μL(1×10 8 cells) with 1 μg transforming DNA and 50 μg single- stranded carrier DNA in microfuge tubes. 7.Add 300 μL sterile plate solution (40 % PEG 4000 + 1×TE/LiAc, for 1 mL plate solution, you should add 0. 8 mL 50 % PEG 4000, 0.1 mL 10×TE, 0.1 mL 10×LiAc). Vortex to mix thoroughly. 8.Incubate at 30 ℃ in the shaker for 30 minutes. 9.Heat shock in a 42 waterbath for 15 minutes(different strains have different optimal heat shock time)

14. Protocol 10.Centrifuge at 13.000 g for 30 s. Remove the supernatant carefully. 11.Resuspend the cell pellet 1.0 mL of 1×TE/sterile water. Stir the pellet with a micropipette tip and vortex. 12.Dilute appropriately and plate on selective medium.