Cultivation and Transformation
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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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Tian He 2008. 07. 06

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Tian he 2008 07 06

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


Tian he 2008 07 06

  • YPD: 1000 mL

  • Yeast Extract 10 g

  • Peptone (Tryptone) 20 g

  • Glucose 20 g

  • Agar (for plates) 20 g

  • Distilled H2O 1000 mL


Tian he 2008 07 06

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!


Tian he 2008 07 06

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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