Yu:Transformation
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Integration
1. Grow yeast cells in YEPD to late-log phase (1-2 x 107 cells/mL) at 30°C | ||
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) | ||
3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes. | ||
4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H2O. Spin down again at 2000 RPM for 2 minutes | ||
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 | ||
7. Discard supernatant, resuspend the pellet in 1mL fresh 1x LiOAc/TE, then spin down at 12000 RPM for 10 seconds again | ||
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 | ||
9. Discard supernatant and resuspend the pellet in 75μL 1x LiOAc/TE | ||
10. Add 5μL boiled ssDNA to protect plasmid DNA from DNAse degredation | ||
11. Add 1μg plasmid DNA or PCR product | ||
12. Incubate at 30°C for 30 minutes on a rocker | ||
13. Add 300μL 40% PEG in LiOAc and vortex thoroughly | ||
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14. Incubate again at 30°C for 30 minutes on a rocker | ||
15. Add 35μL DMSO and heat shock at 42°C for 15 minutes | ||
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 | ||
17. Spin down for 10 seconds at 12000 RPM and discard supernatant | ||
18. Resuspend cells in 200μL TE8 and plate on selective media with glass beads or a spreader |
Plasmid Transformation
1. Grow yeast cells in YEPD or plasmid-maintaining selective media to late-log phase (1-2 x 107 cells/mL) at 30°C |
2. Subculture overnight yeast culture at 1:100 in fresh YEPD or plasmid-maintaining selective media and grow on a rocker for 3-4 hours at 30°C |
3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes. |
4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H2O. Spin down again at 2000 RPM for 2 minutes |
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 |
7. Discard supernatant, resuspend the pellet in 1mL fresh 1x LiOAc/TE, then spin down at 12000 RPM for 10 seconds again |
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 |
9. Discard supernatant and resuspend the pellet in 75μL 1x LiOAc/TE |
10. Add 5μL boiled ssDNA |
11. Add 1μg plasmid DNA |
12. Add 300μL 40% PEG |
13. Incubate at 30°C for 30 minutes |
14. Heat shock at 42°C for 15 minutes |
15. Spin down at 5000 RPM for 1 minute, pipette off supernatant, resuspend in 150μL TE8, and plate onto selective media |