Team:British Columbia/Protocols/Yeast

From 2011.igem.org

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'''Yeast Transformation'''
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==Yeast Transformation==
# Grow overnight culture; rotating/shaking at 30 degrees Celsius  
# Grow overnight culture; rotating/shaking at 30 degrees Celsius  
# The next day, dilute culture 1/10 and grow at 30 degrees Celsius for 3-4 hours
# The next day, dilute culture 1/10 and grow at 30 degrees Celsius for 3-4 hours
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# Pellet and wash once with 0.1M Lithium Acetate (LiAc)
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# Pellet and wash once with 0.1 M Lithium Acetate (LiAc)
# Pellet and discard supernatant
# Pellet and discard supernatant
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# Add in order: 240uL 50% Polyethylene Glycol (PEG), 36uL 1M LiAC, 10uL Boiled Herring Sperm DNA, 50uL of your desired DNA construct (~10ng/uL)
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# Add in order: 240 μL 50% Polyethylene Glycol (PEG), 36 μL 1M LiAC, 10 μL Boiled Herring Sperm DNA, 50 μL of your desired DNA construct (~10 ng/μL)
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# Vortex until uniform and incubate at 30 degrees Celsius for 30min
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# Vortex until uniform and incubate at 30 degrees Celsius for 30 min
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# Heat-shock at 42 degrees Celsius for 20min
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# Heat-shock at 42 degrees Celsius for 20 min
# Pellet and discard supernatant
# Pellet and discard supernatant
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# Resuspend with 200uL sterile water and spread on selective media plate
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# Resuspend with 200 μL sterile water and spread on selective media plate
# Incubate plate at 30 degrees Celsius for 3-4 days and you should see colonies!
# Incubate plate at 30 degrees Celsius for 3-4 days and you should see colonies!
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==Yeast Crude Extract for SDS-PAGE==
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'''Yeast Crude Extract for SDS-PAGE'''
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# Grow overnight culture; rotating/shaking at 30 degrees Celsius  
# Grow overnight culture; rotating/shaking at 30 degrees Celsius  
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# Resuspend in lysis buffer and transfer to capped O-ring tubes
# Resuspend in lysis buffer and transfer to capped O-ring tubes
# Add 200uL of tiny glass beads
# Add 200uL of tiny glass beads
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# Place in lytic beater at 4 degrees Celsius for 7min
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# Place in lytic beater at 4 degrees Celsius for 7 min
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# Centrifuge at 4 degrees Celsius for 10min at 13000rpm
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# Centrifuge at 4 degrees Celsius for 10min at 13000 rpm
# Carefully take the supernatant and transfer to a new microcentrifuge tube
# Carefully take the supernatant and transfer to a new microcentrifuge tube
# Add appropriate amount of 5x SDS-PAGE loading buffer
# Add appropriate amount of 5x SDS-PAGE loading buffer
# Store at -20 degrees Celsius
# Store at -20 degrees Celsius
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'''GFP Fixation (from Koshland Website)'''
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==GFP Fixation for microscopy and fluorescence activated cell sorting (FACS) (from Koshland Website)==
# Pellet yeast cells and discard supernatant
# Pellet yeast cells and discard supernatant
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# Add 100ul of 4% paraformaldehyde, 3.4% sucrose and vortex
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# Add 100 μl of 4% paraformaldehyde, 3.4% sucrose and vortex
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# Incubate at RT for 15min
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# Incubate at RT for 15 min
# Pellet yeast cells and discard supernatant
# Pellet yeast cells and discard supernatant
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# Wash once in KPO4/sorbitol
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# Wash once in KPO4/sorbitol (60 ml 2M sorbitol, 10 ml 1M potassium phosphate, 30 ml water)
# resuspend in small volume of KPO4/sorbitol
# resuspend in small volume of KPO4/sorbitol
# Sonicate cells for 3 seconds on setting 3
# Sonicate cells for 3 seconds on setting 3
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# Pipette one to three microliters of cells onto coverslip
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<html></br></html>
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KPO4/sorbitol
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60 ml 2M sorbitol
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10 ml 1M potassium phosphate
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30 ml water
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Latest revision as of 17:48, 18 October 2011

Team: British Columbia - 2011.igem.org

iGEM Tips for Success
 Things to think about when designing your project and experiments, as well as general safety rules.

Site Directed Mutagenesis
 A molecular biology technique in which a mutation is created at a defined site in a DNA molecule.

Bacterial Standard Operating Protocols
 How to prepare competent cells, transform your construct into competent cells, and express your protein from a lac promoter.

Yeast Standard Operating Protocols
 How to transform your construct into yeast, obtain crude extract for SDS-PAGE, and perform GFP fixation for microscopy and fluorescence-activated cell sorting.

Gas Chromatography-Mass Spectrometry (GC-MS)
 A method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample.

Beetle Transfer Experiments
 Preliminary experiments to probe the efficiency of transferring yeast via beetle vector.

Yeast-Fungi Co-culture Experiments
 Preliminary experiments to investigate the competitive interactions between yeast and the Bluestain Fungus.




Yeast Transformation

  1. Grow overnight culture; rotating/shaking at 30 degrees Celsius
  2. The next day, dilute culture 1/10 and grow at 30 degrees Celsius for 3-4 hours
  3. Pellet and wash once with 0.1 M Lithium Acetate (LiAc)
  4. Pellet and discard supernatant
  5. Add in order: 240 μL 50% Polyethylene Glycol (PEG), 36 μL 1M LiAC, 10 μL Boiled Herring Sperm DNA, 50 μL of your desired DNA construct (~10 ng/μL)
  6. Vortex until uniform and incubate at 30 degrees Celsius for 30 min
  7. Heat-shock at 42 degrees Celsius for 20 min
  8. Pellet and discard supernatant
  9. Resuspend with 200 μL sterile water and spread on selective media plate
  10. Incubate plate at 30 degrees Celsius for 3-4 days and you should see colonies!

Yeast Crude Extract for SDS-PAGE

  1. Grow overnight culture; rotating/shaking at 30 degrees Celsius
  2. The next day, dilute culture 1/10 and grow at 30 degrees Celsius for 3-4 hours
  3. Pellet and wash once with sterile water
  4. Pellet and discard supernatant
  5. Resuspend in lysis buffer and transfer to capped O-ring tubes
  6. Add 200uL of tiny glass beads
  7. Place in lytic beater at 4 degrees Celsius for 7 min
  8. Centrifuge at 4 degrees Celsius for 10min at 13000 rpm
  9. Carefully take the supernatant and transfer to a new microcentrifuge tube
  10. Add appropriate amount of 5x SDS-PAGE loading buffer
  11. Store at -20 degrees Celsius

GFP Fixation for microscopy and fluorescence activated cell sorting (FACS) (from Koshland Website)

  1. Pellet yeast cells and discard supernatant
  2. Add 100 μl of 4% paraformaldehyde, 3.4% sucrose and vortex
  3. Incubate at RT for 15 min
  4. Pellet yeast cells and discard supernatant
  5. Wash once in KPO4/sorbitol (60 ml 2M sorbitol, 10 ml 1M potassium phosphate, 30 ml water)
  6. resuspend in small volume of KPO4/sorbitol
  7. Sonicate cells for 3 seconds on setting 3


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