Tian He

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

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

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

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!

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!

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

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

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

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

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.

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 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 g and discard the supernatant.

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 ×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)

Protocol 10.Centrifuge at 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.