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

• Glucose 20 g

• Agar (for plates) 20 g

• Distilled H2O 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 H2O 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 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.

pGREG 503/4/5

Autonomously replicating sequence

Auxotroph marker

Antibiotic markerAntibiotic 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 1-2×107/mL(nearly saturated). A suspension containing 1×106 cells/mL gives an OD600 of 0.1.

2. 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.

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.

Protocol

5. Wash cells in 1.0 mL of TE/LiAc(10×) and resuspend at 2×109 cells/mL in TE/LiAc (1×)

6. Mix 50 μL(1×108 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)

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.