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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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Presentation Transcript
slide1

Cultivation and Transformation

of

Yeasts

Tian He

2008. 07. 06

media
Media

non-selective:

YPD without antibiotics

Selective:

Synthetic complete dropout medium (SC)

(auxotroph)

YPD with antibiotics

slide3

YPD: 1000 mL

  • Yeast Extract 10 g
  • Peptone (Tryptone) 20 g
  • Glucose 20 g
  • Agar (for plates) 20 g
  • Distilled H2O 1000 mL
slide4
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
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
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!
slide7
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
pGREG 503/4/5

Homologous sequence

Tag

Promoter

Antibiotic marker

Antibiotic marker

Stuffer

Auxotroph marker

Autonomously replicating sequence

yeast strain ah109
Yeast strain: AH109

GAL 4 induced promoters: GAL1, GAL2, MEL1

transformation
Transformation

Plasmid/ssDNA/dsDNA

The competent cells of yeast cannot be stored;

it must be prepared before use!

materials
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
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.

protocol1
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)
protocol2
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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