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Yeast Transformation II

Low Efficiency Transformation (for Plasmids)

Inoculate 5 mls per transformation. Grow yeast overnight (use YPD if possible, selective media if necessary).

High Efficiency (for Linear DNA or Libraries)

Inoculate 5 mls. Grow yeast overnight. Inoculate 50 mls YPD to OD600 0.07. Grow for 4-5 doubles (OD600 0.6-1.0).

For Both

  1. Harvest cells. Spin at 2500 rpm for 5 minutes, and discard media.
  2. Wash once with sterile H2O.
  3. Resuspend in 1-3 mls sterile H2O, transfer to Eppendorf tube, spin down, discard supernatant.
  4. Resuspend in 1X lithium acetate/TE, spin down, discard supernatant.
  5. Add ~1 µg (in 1-8 µl) transforming DNA and 10 µl of single stranded carrier DNA. Mix gently.
  6. Add 600 µl PEG/lithium acetate/TE solution, and mix gently.
  7. Incubate with gentle rotation at 30°C for 30 min.
  8. Heat shock at 42°C for 15 min.
  9. Add sterile H2O, and wash yeast 2-3 times.
  10. Resuspend in 500 µl of H2O.
  11. Plate on appropriate selective media.

Solutions

Carrier DNA

  • 10 mg/ml
  • Boil 5 min
  • Place on ice for 5 min before use

10X Lithium Acetate

  • 1M, pH 7.5

10X TE

  • 100mM Tris, pH 7.5
  • 10mM EDTA
  • Filter sterilize

50% PEG (3500-4000)

  • Dissolve in water
  • May have to heat to dissolve

1X Lithium Acetate/TE

  • 100 µl 10X TE
  • 100 µl 10X LiAc
  • 800 µl H2O

PEG/Lithium Acetate/TE

  • 960 µl 50% PEG
  • 120 µl 10X LiAc
  • 120 µl 10X TE

© 2014 The University of Texas MD Anderson Cancer Center