| 1. Grow yeast cells in YEPD to late-log phase (1-2 x 107 cells/mL) at 30°C
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| 2. Subculture overnight yeast culture at 1:100 in fresh YEPD and grow to mid-log phase (OD600≈0.6, approximately 3-4 hours at 30°C for most strains)
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| 3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes.
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| 4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H2O. Spin down again at 2000 RPM for 2 minutes
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| 5. Discard H2O supernatant and resuspend pellet in 1mL sterile H2O. Transfer to a 1.5mL sterile eppendorf tube and spin down at 12000 RPM for 10 seconds
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| 7. Discard supernatant, resuspend the pellet in 1mL fresh 1x LiOAc/TE, then spin down at 12000 RPM for 10 seconds again
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| 8. Discard supernatant, resuspend the pellet in 1mL fresh 1x LiOAc/TE a second time, then spin down at 12000 RPM for 10 seconds again
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| 9. Discard supernatant and resuspend the pellet in 75μL 1x LiOAc/TE
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| 10. Add 5μL boiled ssDNA to protect plasmid DNA from DNAse degredation
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| 11. Add 1μg plasmid DNA or PCR product
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| 12. Incubate at 30°C for 30 minutes on a rocker
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| 13. Add 300μL 40% PEG in LiOAc and vortex thoroughly
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300μL 40% PEG in LiOAc/TE
| 240μL 50% PEG (MW 3350), sterile filtered
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| 60μL 5x LiOAc/TE. Do not use 1x LiOAc/TE
|
|
| 14. Incubate again at 30°C for 30 minutes on a rocker
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| 15. Add 35μL DMSO and heat shock at 42°C for 15 minutes
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| 16. For antimicrobial screening, such as G418 or CLONAT, add the entire mixture to 5mL of YEPD and incubate at 30°C for 3 hours to allow recovery and the production of resistance proteins. Otherwise, if screening with amino acid synthesis, like His+ or Leu+, skip this step
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| 17. Spin down for 10 seconds at 12000 RPM and discard supernatant
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| 18. Resuspend cells in 200μL TE8 and plate on selective media with glass beads or a spreader
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