Yu:Transformation

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|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
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|valign="top"|Wash in 1mL 1x LiOAc/TE
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|valign="top"|Wash in 1mL 1x [[Yu:LiOAc|LiOAc/TE]]
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|valign="top"|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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|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
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|valign="top"|Wash a second time in 1mL 1x LiOAc/TE
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|valign="top"|Wash a second time in 1mL 1x [[Yu:LiOAc|LiOAc/TE]]
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|valign="top"|9. Discard supernatant and resuspend the pellet in 75μL 1x LiOAc/TE
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|valign="top"|9. Discard supernatant and resuspend the pellet in 75μL 1x [[Yu:LiOAc|LiOAc/TE]]
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|valign="top"|Resuspend in 75μL 1x LiOAc/TE
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|valign="top"|Resuspend in 75μL 1x [[Yu:LiOAc|LiOAc/TE]]
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|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

Revision as of 09:48, 15 September 2009

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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 Grow yeast
2. Subculture 100mL of yeast culture into 10mL YEPD and grow on a rocker for 3-4 hours at 30°C Subculture 100mL 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
300μL 40% PEG in LiOAc/TE
240μL 50% PEG, sterile filtered
60μL 5x LiOAc/TE. Do not use 1x LiOAc/TE
14. Incubate again at 30°C for 30 minutes on a rocker Incubate 30°C for 30 minutes
15. Add 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
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