Yu:Transformation
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1. Grow yeast cells in YEPD to late-log phase (1-2 x 107 cells/mL) at 30°C | Grow yeast | ||
2. Subculture overnight yeast culture at 1:100 in fresh YEPD and grow on a rocker for 3-4 hours at 30°C | Subculture 100μL into 10mL YEPD and grow for 3-4 hours | ||
3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes. | Spin down at 2k RPM for 2 minutes | ||
4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H2O. Spin down again at 2000 RPM for 2 minutes | Discard supernatant and wash in 10mL sterile water | ||
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 | Discard supernatant, resuspend in 1mL sterile H2O in a 1.5mL tube, and spin down | ||
7. Discard supernatant, resuspend the pellet in 1mL fresh 1x LiOAc/TE, then spin down at 12000 RPM for 10 seconds again | Wash in 1mL 1x LiOAc/TE | ||
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 | Wash a second time in 1mL 1x LiOAc/TE | ||
9. Discard supernatant and resuspend the pellet in 75μL 1x LiOAc/TE | Resuspend in 75μL 1x LiOAc/TE | ||
10. Add 5μL boiled ssDNA to protect plasmid DNA from DNAse degredation | Add 5μL boiled ssDNA | ||
11. Add 1μg plasmid DNA or PCR product | Add DNA | ||
12. Incubate at 30°C for 30 minutes on a rocker | Incubate 30°C for 30 minutes | ||
13. Add 300μL 40% PEG in LiOAc and vortex thoroughly | Add 300μL 40% PEG | ||
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14. Incubate again at 30°C for 30 minutes on a rocker | Incubate 30°C for 30 minutes | ||
15. AdμLd 35μL DMSO and heat shock at 42°C for 15 minutes | Add 35μL DMSO, heat shock 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 | Recover in 5mL YEPD at 30°C for 3 hours if needed | ||
17. Spin down for 10 seconds at 12000 RPM and discard supernatant | Spin down and discard supernatant | ||
18. Resuspend cells in 200μL TE8 and plate on selective media with glass beads or a spreader | Resuspend in 200μL TE8 and plate |