1x Scalable Yeast Transformation Protocol

*after Agatep et al., 1999 (KA Longo, 1/01)

• 1. Inoculated 25 ml of SD/Trp- media with ~10 colonies of Yeas strain 2.3 expressing pAS2-1/expCR4.1, and incubated overnight @ 30°C, 250rpm.

  2. Perform a cell count on the overnight growth, and dilute the cells in 50 ml of pre-warmed (30°C) YPAD to a final concentration of 5 x 106 cells/ml.

  3. Incubate @ 30°C until the culture density has reached 2 x 107 cells/ml (~3-4 h).

  4. Spin the cells for 5 min @ 3000 x g. Discard supernatant.

  5. Resuspend cells in 25 ml sterile ddH2O. Repeat step 4.

  6. Resuspend the pellet in 900 ml sterile ddH2O and transfer the cells to a sterile microfuge tube.

  7. Pellet cells @ 13,000 rpm for 1 min. discard supernatant.

  8. Resuspend cells in enough 100 mM LiAc (pH 7.5) to give a final volume of 1 ml.

  9. Incubate @ 30°C, 10 min.

  10. Aliquot 100 ml of cell suspension into several microfuge tubes, and pellet @ 13,000 rpm for 1 min. Discard the supernatant.

  11. Add the following to the pellets in the order stipulated:

• 1. Vortex vigorously until the pellet is completely resuspended.

  2. Incubate transformation mixtures for 30 min @ 30°C without shaking.

  3. Heat shock cells for 30 min @ 42°C.

  4. Pellet cells @13,000 rpm for 1 min and remove supernatant.

  5. Add 1 ml sterile ddH2O to cells, and resuspend the cells by gentle pipetting. A brief vortex may be necessary to completely dissolve the pellet before plating.

  6. Plate 100 ml, 10 ml, and 10 ml on each of three SD/Trp-/Leu- agar plates. Incubate for 3-4 days until colonies appear.