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Tian He 2008. 07. 06

Cultivation and Transformation of Yeasts. Tian He 2008. 07. 06. 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

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Tian He 2008. 07. 06

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  1. Cultivation and Transformation of Yeasts 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 H2O 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 H2O 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 Homologous sequence Tag Promoter Antibiotic marker Antibiotic marker Stuffer Auxotroph marker Autonomously replicating sequence

  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 • Inoculate cells into 50 mL YPD and grow overnight to a density of 1-2×107/mL(nearly saturated). A suspension containing 1×106 cells/mL gives an OD600 of 0.1. • Dilute to 2×106 /mL in fresh YPD and re-grow into exponential phase (1×107/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. • Harvest the culture in a sterile centrifuge tube at 2500 rpm for 5 minutes. Wash in sterile water twice. • 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 • Wash cells in 1.0 mL of TE/LiAc(10×) and resuspend at 2×109 cells/mL in TE/LiAc (1×) • Mix 50 μL(1×108 cells) with 1 μg transforming DNA and 50 μg single-stranded carrier DNA in microfuge tubes. • 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. • Incubate at 30℃ in the shaker for 30 minutes. • Heat shock in a 42 waterbath for 15 minutes(different strains have different optimal heat shock time)

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

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