*after Agatep et al., 1999 (KA Longo, 1/01)
- 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.
- 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.
- Incubate @ 30°C until the culture density has reached 2 x 107 cells/ml (~3-4 h).
- Spin the cells for 5 min @ 3000 x g. Discard supernatant.
- Resuspend cells in 25 ml sterile ddH2O. Repeat step 4.
- Resuspend the pellet in 900 ml sterile ddH2O and transfer the cells to a sterile microfuge tube.
- Pellet cells @ 13,000 rpm for 1 min. discard supernatant.
- Resuspend cells in enough 100 mM LiAc (pH 7.5) to give a final volume of 1 ml.
- Incubate @ 30°C, 10 min.
- Aliquot 100 ml of cell suspension into several microfuge tubes, and pellet @ 13,000 rpm for 1 min. Discard the supernatant.
- Add the following to the pellets in the order stipulated:
| 50 % PEG | 240 µL | | 1.0 M LiAc, pH 7.5 | 36 µL | | ss-DNA (2 mg/mL) | 50 µL | | Plasmid DNA | X µL | | Sterile ddH2O | X µL | | FINAL VOLUME | 360 µL |
- Vortex vigorously until the pellet is completely resuspended.
- Incubate transformation mixtures for 30 min @ 30°C without shaking.
- Heat shock cells for 30 min @ 42°C.
- Pellet cells @13,000 rpm for 1 min and remove supernatant.
- 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.
- 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.
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