Yu:Transformation: Difference between revisions

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|+'''High-Efficienty Lithium Acetate Transformation for ''S. cerevisiae'''''
 
|width="600px"|1. Grow yeast cells in [[Yu:YEPD|YEPD]] to late-log phase (1-2 x 10<sup>7</sup> cells/mL) at 30°C
===Integration===
|width="200px"|Grow yeast
{| 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 100mL of yeast culture into 10mL 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 to mid-log phase (OD<sub>600</sub>≈0.6, approximately 3-4 hours at 30°C for most strains)
|valign="top"|Subculture 100mL into 10mL YEPD and grow for 3-4 hours
|-
|-
|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"|Spin down at 2k 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"|Discard supernatant and wash in 10mL sterile water
|-
|-
|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"|Discard supernatant, resuspend in 1mL sterile H<sub>2</sub>O in a 1.5mL tube, and spin down
|-
|-
|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"|Wash in 1mL 1x [[Yu:LiOAc|LiOAc/TE]]
|-
|-
|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"|Wash a second time in 1mL 1x [[Yu:LiOAc|LiOAc/TE]]
|-
|-
|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"|Resuspend 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"|Add 5μL boiled ssDNA
|-
|-
|valign="top"|11. Add 1μg plasmid DNA or PCR product
|valign="top"|11. Add 1μg plasmid DNA or PCR product
|valign="top"|Add DNA
|-
|-
|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"|Incubate 30°C for 30 minutes
|-
|-
|valign="top"|13. Add 300μL 40% PEG in LiOAc and vortex thoroughly
|valign="top"|13. Add 300μL 40% PEG in LiOAc and vortex thoroughly
|valign="top"|Add 300μL 40% PEG
|-
|-
|colspan="2" align="center"|
|align="center"|
{|border="0" cellpadding="5"
{|border="1"  
|+ '''300μL 40% PEG in LiOAc/TE'''
|+'''300μL 40% PEG in LiOAc/TE'''
|240μL 50% PEG, sterile filtered
|-
|-
|60μL 5x [[Yu:LiOAc|LiOAc/TE]]. Do not use 1x LiOAc/TE
|align="center"|240μL 50% PEG (MW 3350), 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"|Incubate 30°C for 30 minutes
|-
|-
|valign="top"|15. Add 35μL DMSO and heat shock at 42°C for 15 minutes
|valign="top"|15. Add 35μL DMSO and heat shock at 42°C for 15 minutes
|valign="top"|Add 35μL DMSO, heat shock 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"|Recover in 5mL YEPD at 30°C for 3 hours if needed
|-
|-
|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"|Spin down 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"|Resuspend in 200μL TE<sub>8</sub> and plate
|}
===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
|}

Latest revision as of 14:21, 4 May 2010

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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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