Yu:Transformation

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Contents


Integration

High-Efficienty Lithium Acetate Transformation for S. cerevisiae
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
300μL 40% PEG in LiOAc/TE
240μL 50% PEG (MW 3350), sterile filtered
60μL 5x LiOAc/TE. Do not use 1x LiOAc/TE
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

High-Efficienty Lithium Acetate Plasmid Transformation for S. cerevisiae
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
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