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Yeast Transformation I

Method

  1. Grow a 25 to 50 ml culture of yeast to log phase, OD600 of 0.6 to 2.0. You need about 10-20 ml of culture for each transformation.

    OD is fairly flexible, but log phase will give the highest transformation efficiency. If you are transforming a supercoiled plasmid – say from a maxi-prep – then even yeast scraped off a fresh plate will work ('fresh' means just grown, not stored in the refrigerator yet).

    If you are trying to do a knock-out or have a small amount of DNA or impure DNA, then pay special attention to getting the yeast at the correct density. In this lab, we usually grow a small overnight culture (2-5 ml) and then seed a larger culture in the morning. Use 1-2 ml culture for each 50 ml media seeded. Let grow at least 4 hours.

  2. Spin culture down in a 50-ml tube. Discard supernatant.
  3. Wash yeast pellet in 10 ml of Li-TE. Spin. Discard supernatant.
  4. Resuspend pellet in Li-TE. Use 100 µl per 10-ml culture. For example, use 1 ml for 100 ml of original culture.
  5. Put 5 µl of DNA to be transformed and 5 µl of carrier DNA (calf thymus or salmon sperm DNA, 10 mg/ml) into a sterile microfuge tube. Add 100 µl of yeast and tap to mix.
  6. Let sit at room temperature for 5 minutes.
  7. Add 280 µl of 40% PEG in Li-TE to the transformation and invert to mix.
  8. Incubate 45 minutes at 30°C.
  9. Add 45 µl of DMSO, mix by inversion and heat shock for 5 minutes at 42°C.
  10. Pellet yeast with short microfuge spin.
  11. Aspirate supernatant. Change to a new sterile tip for each sample.
  12. Resuspend yeast in 1 ml of sterile H2O. Spin and discard supernatant as in step 11.
  13. Resuspend yeast pellet in 0.5 ml of sterile H2O and plate on appropriate selective media.
  14. Incubate 2-4 days at 30°C.

Solutions

1 M Lithium Acetate

  • Filter sterilize.

10X TE

(filter sterilized)

  • 10 ml 1M Tris, pH 8.0
  • 2 ml 0.5M EDTA
  • 88 ml H20

Li-TE

  • 10 ml 1M Lithium Acetate
  • 10 ml 10X TE
  • 80 ml H2O
  • Filter sterilize

40% PEG

  • Make 50% PEG in H20
  • Autoclave.
  • Add 1/10 volume 1M lithium acetate
  • Add 1/10 volume 10X TE

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