Yu:Transformation: Difference between revisions
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{| border="0" cellpadding="5" | __TOC__ | ||
|+'''High-Efficienty Lithium Acetate Transformation for | |||
| | ===Integration=== | ||
{| border="0" cellpadding="5" | |||
|+ '''''High-Efficienty Lithium Acetate Transformation for S. cerevisiae''''' | |||
|valign="top"|1. Grow yeast cells in [[Yu:YEPD|YEPD]] to late-log phase (1-2 x 10<sup>7</sup> cells/mL) at 30°C | |||
|- | |- | ||
|valign="top"|2. Subculture overnight yeast culture at 1:100 in fresh YEPD and grow on a rocker for 3-4 hours at 30°C | |valign="top"|2. Subculture overnight yeast culture at 1:100 in fresh YEPD and grow on a rocker for 3-4 hours at 30°C | ||
|- | |- | ||
|valign="top"|3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes. | |valign="top"|3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes. | ||
|- | |- | ||
|valign="top"|4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H<sub>2</sub>O. Spin down again at 2000 RPM for 2 minutes | |valign="top"|4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H<sub>2</sub>O. Spin down again at 2000 RPM for 2 minutes | ||
|- | |- | ||
|valign="top"|5. Discard H<sub>2</sub>O supernatant and resuspend pellet in 1mL sterile H<sub>2</sub>O. Transfer to a 1.5mL sterile eppendorf tube and spin down at 12000 RPM for 10 seconds | |valign="top"|5. Discard H<sub>2</sub>O supernatant and resuspend pellet in 1mL sterile H<sub>2</sub>O. Transfer to a 1.5mL sterile eppendorf tube and spin down at 12000 RPM for 10 seconds | ||
|- | |- | ||
|valign="top"|7. Discard supernatant, resuspend the pellet in 1mL fresh 1x [[Yu:LiOAc|LiOAc/TE]], then spin down at 12000 RPM for 10 seconds again | |valign="top"|7. Discard supernatant, resuspend the pellet in 1mL fresh 1x [[Yu:LiOAc|LiOAc/TE]], then spin down at 12000 RPM for 10 seconds again | ||
|- | |- | ||
|valign="top"|8. Discard supernatant, resuspend the pellet in 1mL fresh 1x [[Yu:LiOAc|LiOAc/TE]] a second time, then spin down at 12000 RPM for 10 seconds again | |valign="top"|8. Discard supernatant, resuspend the pellet in 1mL fresh 1x [[Yu:LiOAc|LiOAc/TE]] a second time, then spin down at 12000 RPM for 10 seconds again | ||
|- | |- | ||
|valign="top"|9. Discard supernatant and resuspend the pellet in 75μL 1x [[Yu:LiOAc|LiOAc/TE]] | |valign="top"|9. Discard supernatant and resuspend the pellet in 75μL 1x [[Yu:LiOAc|LiOAc/TE]] | ||
|- | |- | ||
|valign="top"|10. Add 5μL boiled ssDNA to protect plasmid DNA from DNAse degredation | |valign="top"|10. Add 5μL boiled ssDNA to protect plasmid DNA from DNAse degredation | ||
|- | |- | ||
|valign="top"|11. Add 1μg plasmid DNA or PCR product | |valign="top"|11. Add 1μg plasmid DNA or PCR product | ||
|- | |- | ||
|valign="top"|12. Incubate at 30°C for 30 minutes on a rocker | |valign="top"|12. Incubate at 30°C for 30 minutes on a rocker | ||
|- | |- | ||
|valign="top"|13. Add 300μL 40% PEG in LiOAc and vortex thoroughly | |valign="top"|13. Add 300μL 40% PEG in LiOAc and vortex thoroughly | ||
|- | |- | ||
| | |align="center"| | ||
{|border=" | {|border="1" | ||
|+ '''300μL 40% PEG in LiOAc/TE''' | |+'''300μL 40% PEG in LiOAc/TE''' | ||
|- | |- | ||
|60μL 5x [[Yu:LiOAc|LiOAc/TE]]. Do not use 1x LiOAc/TE | |align="center"|240μL 50% PEG, sterile filtered | ||
|- | |||
|align="center"|60μL 5x [[Yu:LiOAc|LiOAc/TE]]. Do not use 1x LiOAc/TE | |||
|} | |} | ||
|- | |- | ||
|align="left" valign="top"|14. Incubate again at 30°C for 30 minutes on a rocker | |align="left" valign="top"|14. Incubate again at 30°C for 30 minutes on a rocker | ||
|- | |- | ||
|valign="top"|15. | |valign="top"|15. Add 35μL DMSO and heat shock at 42°C for 15 minutes | ||
|- | |- | ||
|valign="top"|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<sup>+</sup> or Leu<sup>+</sup>, skip this step | |valign="top"|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<sup>+</sup> or Leu<sup>+</sup>, skip this step | ||
|- | |- | ||
|valign="top"|17. Spin down for 10 seconds at 12000 RPM and discard supernatant | |valign="top"|17. Spin down for 10 seconds at 12000 RPM and discard supernatant | ||
|- | |- | ||
|valign="top"|18. Resuspend cells in 200μL TE<sub>8</sub> and plate on selective media with glass beads or a spreader | |valign="top"|18. Resuspend cells in 200μL TE<sub>8</sub> and plate on selective media with glass beads or a spreader | ||
|valign="top"| | |} | ||
===Plasmid Transformation=== | |||
{| border="0" cellpadding="5" | |||
|+ '''''High-Efficienty Lithium Acetate Plasmid Transformation for S. cerevisiae''''' | |||
|valign="top"|1. Grow yeast cells in [[Yu:YEPD|YEPD]] or plasmid-maintaining selective media to late-log phase (1-2 x 10<sup>7</sup> cells/mL) at 30°C | |||
|- | |||
|valign="top"|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 | |||
|- | |||
|valign="top"|3. Transfer the 10mL yeast culture into a 15mL tube and spin down at 2000 RPM for 2 minutes. | |||
|- | |||
|valign="top"|4. Discard YEPD supernatant and resuspend pellet in 10mL sterile H<sub>2</sub>O. Spin down again at 2000 RPM for 2 minutes | |||
|- | |||
|valign="top"|5. Discard H<sub>2</sub>O supernatant and resuspend pellet in 1mL sterile H<sub>2</sub>O. Transfer to a 1.5mL sterile eppendorf tube and spin down at 12000 RPM for 10 seconds | |||
|- | |||
|valign="top"|7. Discard supernatant, resuspend the pellet in 1mL fresh 1x [[Yu:LiOAc|LiOAc/TE]], then spin down at 12000 RPM for 10 seconds again | |||
|- | |||
|valign="top"|8. Discard supernatant, resuspend the pellet in 1mL fresh 1x [[Yu:LiOAc|LiOAc/TE]] a second time, then spin down at 12000 RPM for 10 seconds again | |||
|- | |||
|valign="top"|9. Discard supernatant and resuspend the pellet in 75μL 1x [[Yu:LiOAc|LiOAc/TE]] | |||
|- | |||
|valign="top"|10. Add 5μL boiled ssDNA | |||
|- | |||
|valign="top"|11. Add 1μg plasmid DNA | |||
|- | |||
|valign="top"|12. Add 300μL 40% PEG | |||
|- | |||
|valign="top"|13. Incubate at 30°C for 30 minutes | |||
|- | |||
|valign="top"|14. Heat shock at 42°C for 15 minutes | |||
|- | |||
|valign="top"|15. Spin down at 5000 RPM for 1 minute, pipette off supernatant, resuspend in 150μL TE<sub>8</sub>, and plate onto selective media | |||
|} |
Revision as of 15:05, 26 February 2010
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 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 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 | ||
| ||
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 |