Team:British Columbia/Protocols/Gcms

From 2011.igem.org

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{{Template:Protocols}}
{{Template:Protocols}}
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'''GC-MS Sample Preparation Protocol for yeast'''
 
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Supplies Needed:
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 +
 
 +
==GC-MS Sample Preparation Protocol for yeast==
 +
 
 +
 
 +
'''Supplies Needed:'''
# Erlenmeyer Flasks
# Erlenmeyer Flasks
# Appropriate Media [Glucose (GPD) or Galactose (GAL)]
# Appropriate Media [Glucose (GPD) or Galactose (GAL)]
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# 5mL Pasteur Pipets and Bulbs
# 5mL Pasteur Pipets and Bulbs
-
Steps:
+
'''Procedures:'''
Part 1: Preparation for Extraction
Part 1: Preparation for Extraction
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# Make 5mL overnight cultures of yeasts to be sampled (18-20 hours@ 30 degrees). The media used here must be SD-Leu (glucose).
+
# Make 5 mL overnight cultures of yeasts to be sampled (18-20 hours @ 30 degrees). The media used here must be SD-Leu (glucose).
-
# Measure OD600 of the cultures using the spectrophotometer. The OD600 should be around 2.
+
# Measure OD 600 of the cultures using the spectrophotometer. The OD 600 should be around 2.
-
# Add the remaining cultures to Erlenmeyer flasks and dilute with 50mL of of SD-Leu.
+
# Add the remaining cultures to Erlenmeyer flasks and dilute with 50 mL of of SD-Leu.
-
# Grow up the cultures for about 2-3 hours (until OD600 is 0.6-0.8).
+
# Grow up the cultures for about 2-3 hours (until OD 600 is 0.6-0.8).
-
# Transfer all of the cultures into falcon tubes and spin down the cells (5 min@ 2500g).
+
# Transfer all of the cultures into falcon tubes and spin down the cells (5 min @ 2500g).
# Pour out media and resuspend the yeast pellets with about 47mL of SD-Leu (GPD) or SG-Leu (GAL).
# Pour out media and resuspend the yeast pellets with about 47mL of SD-Leu (GPD) or SG-Leu (GAL).
# Transfer the cultures to Erlenmeyer flasks and grow up overnight (18-20 hours@ 30 degrees).
# Transfer the cultures to Erlenmeyer flasks and grow up overnight (18-20 hours@ 30 degrees).
# Harvest cells (step 5).
# Harvest cells (step 5).
# Weigh the large glass tubes and record values.
# Weigh the large glass tubes and record values.
-
# Keep 1mL of the media and transfer to large glass tubes and discard the rest of the media.
+
# Keep 1 mL of the media and transfer to large glass tubes and discard the rest of the media.
-
# Wash the yeast pellet with 5mL of sdH2O.
+
# Wash the yeast pellet with 5 mL of sdH2O.
# Transfer the 5mL of yeast to the glass tubes.
# Transfer the 5mL of yeast to the glass tubes.
-
# Spin down the cells (5 min @ 2500g).
+
# Spin down the cells (5 min @ 2500 x g).
# Pour out water and weigh the glass tubes again to obtain the weight of the yeast pellet.
# Pour out water and weigh the glass tubes again to obtain the weight of the yeast pellet.
# Proceed to Extraction.
# Proceed to Extraction.
Line 38: Line 42:
Part 2: Extraction (all of the following steps should be done in the fumehood in Bohlmann lab)
Part 2: Extraction (all of the following steps should be done in the fumehood in Bohlmann lab)
-
# Pipet in 2mL of pentane to each glass tube.
+
# Pipet in 2 mL of pentane to each glass tube.
-
# Add 0.5-1mL of glass beads to each tube.
+
# Add 0.5-1 mL of glass beads to each tube.
# Vortex as fast as possible without spilling for about 20 seconds.
# Vortex as fast as possible without spilling for about 20 seconds.
# Pour solvent into smaller glass tube. Pipet the solvent out for the media samples using the pasteur pipets. Avoid glass beads!
# Pour solvent into smaller glass tube. Pipet the solvent out for the media samples using the pasteur pipets. Avoid glass beads!
-
# Add about 200uL of Sodium Sulphate to the small glass tubes.
+
# Add about 200 μL of Sodium Sulphate to the small glass tubes.
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# Make one blank (4mL of pentane and Sodium Sulphate).
+
# Make one blank (4 mL of pentane and Sodium Sulphate).
# Repeat 1-4.
# Repeat 1-4.
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# Bring down the volume to about 1mL by applying a stream of nitrogen.
+
# Bring down the volume to about 1 mL by applying a stream of nitrogen.
# Place GC vials in a rack and label them.
# Place GC vials in a rack and label them.
# Pipet out solvent and transfer to vials.
# Pipet out solvent and transfer to vials.
-
# Apply nitrogen to vials and bring down the volume to about 0.4mL.
+
# Apply nitrogen to vials and bring down the volume to about 0.4 mL.
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# Add pentane to 0.5mL line.
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# Add pentane to 0.5 mL line.
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'''Protocol for bacteria'''
+
==GC-MS Sample Preparation Protocol for bacteria==
 +
 
 +
'''Supplies Needed:'''
 +
# Erlenmeyer Flasks
 +
# Appropriate Media [LB Broth and Terrific Broth)
 +
# Spectrophotometer         
 +
# sdH2O
 +
# Falcon Tubes
 +
# GLASS test tubes (large and small)
 +
# GC Vials
 +
# 5mL Pasteur Pipets and Bulbs
 +
# His-tag protein purification column
 +
# A chem stand with a clamp
 +
# Large centrifuge tubes
 +
# Nanodrop
 +
 
 +
'''Reagents Needed:'''
 +
# Lysis Buffer
 +
# Wash Buffer
 +
# Elution Buffer
 +
# 0.5M NaOH
 +
# GPP (geranyl pyrophosphate)
 +
# Enzyme Assay Buffer
 +
# Pentane
 +
# DTT (final conc of 5 mM)
 +
# Protein Inhibitory Cocktail (PIC)
 +
 +
'''Procedures:'''
 +
 
 +
Part 1: Preparation for Extraction
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Protein expression of limonene synthase
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# Make 50 mL overnight cultures of selected colony to be sampled (18-20 hours @ 37 degrees). The media used here must be LB broth with the selectable antibiotics.
 +
# Transfer 1 mL of overnight culture (in LB broth) to 250 mL of Terrific broth with the selectable antibiotics in a 1 L or 2 L Erlenmeyer flask.
 +
# Grow up the cultures for about 4-5 hours @ 37 degrees (until OD600 is 0.8).
 +
# Induce cells with 250 μL of 1M IPTG. Incubate culture for 16-18 hours at 16 degrees, shaking at 205 rpm.
 +
# Pour the culture into the centrifuge tubes.
 +
# Spin cells at 3000 rpm at 4 degrees for 20 minutes.
 +
# Transfer the cultures to Erlenmeyer flasks and grow up overnight (18-20 hours @ 30 degrees).
 +
# Carefully pour out supernatent and keep the pellet
 +
# Proceed to extraction or keep pellet at -80 degrees
-
#1. Grow 5 ml culture in LB overnight with appropriate antibiotics
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Part 2: Extraction
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#2. Pour 500 ml of 'terrific broth' into a 2 litre flask
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# Set up stand and clamp with the his-tag purification column. (Note: His-tag Column must never run dry. Therefore, it is advantageous to keep the column wet with wash buffer)
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#3. Add appropriate antibiotics
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# Add 1 tablet of protease inhibitor cocktail and DTT (final conc of 5 mM) to 50 mL of Lysis Buffer
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#4. Pipette 1 ml of culture into 500 mL
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# Weigh out 1-1.5 g of cell pellet into falcon tube
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#5. Grow to OD600 = 0.8
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# Add 5 mL of Lysis Buffer (containing protease inhibitor and TPP) to the cell pellet. Pipet up and down until the pellet is dissolved and the cell lysate is homogeneous.
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#6. Add IPTG to final concentration of 1mM to 500 mL flask
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# Sonicate cells (ready when there's a change in color; usually a lighter colour than the cell pellet)
-
#7. Induce expression at 16 degrees at 200 rpm
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# Centrifuge sonicated cells at max speed for > 20 min (until all cells are pelleted)
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#8. Induce expression over night
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# Collect supernatent (should be around 5 mL).
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#10. Spin down cells in centrifuge tubes, discard supernatant
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# Transfer supernatent into the his-tag column. Adjust flow such that the drop of supernatent passing the column is about 1-2 drops per second. Supernatent can be passed through column twice.
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#11. Store pellets at -80
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# Add 10 mL of wash buffer. Collect drops slightly after the start of wash buffer flow through the column. Nanodrop to find out the absorbance and concentration of proteins passing through the column. Once this 10 mL of wash buffer has passed through, add another 10 mL of wash buffer to wash the column again to collect any residue proteins not binding to the beads of the column. Take a nanodrop reading at the end of the washing steps (last few drops of the wash buffer). Nanodrop reading should read 0 mg/mL at the end of second wash. If proteins are still present, continue with another wash until no protein can be found in the wash buffer.
 +
# Elute with 5mL of elution buffer and collect into a falcon tube.
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Extraction and preparation
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Part 3: GPP assay
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#1. Prepare assay buffer (see below)
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# Pipet in 1 mL of purified protein to 4mL of enzyme assay buffer in a test tube
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#2. Re-suspend 1-2 grams of pellet per 4 ml of assay buffer in a falcon tube
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# Add 7 μL of GPP
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#3. Sonicate cells until pellet turns translucent
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# Add 1-2 mL of pentane (less is better in terms of getting high concentration yield; but may need large volume of pentane to avoid emulsion formation)
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#4. Spin down cells at max speed
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# Incubate in water bath for 1 hour at 30 degrees celsius
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#5. Pipette out supernatant (contains the limonene synthase) into another falcon tube
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# Place GC vial in a rack and label it.
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#6. Discard cell pellets
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# Pipet out solvent (the less dense pentane layer) and transfer to vials to 0.5 mL line.
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#7. Take 2 ml of supernatant and pipette into a different tube
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# Prepare a control without GPP added
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#8. Add geranyl diphosphate (GPP) to a final concentration of 20 uM to the supernatant
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# Freeze both samples
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#9. Add 1 ml of pentane (with isobutylbenzene) to the supernatant
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# Send for GC-MS
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#10. Incubate at 30 degrees for 1 hr
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#11. Pipette out pentane layer into a glass tube with a pasteur pipette
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-
#12. Add another 2 ml of pentane to supernatant
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-
#13. Pipette out the 2 ml of pentane into the glass tube with a pasteur pipette
+
-
#14. Evaporate the total pentane to a final volume of 500 uL using nitrogen gas
+
-
#15. Pipette 500 ul into an agilent vial
+
-
#16. Send to Lina
+

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.




GC-MS Sample Preparation Protocol for yeast

Supplies Needed:

  1. Erlenmeyer Flasks
  2. Appropriate Media [Glucose (GPD) or Galactose (GAL)]
  3. Spectrophotometer
  4. sdH2O
  5. Falcon Tubes
  6. GLASS test tubes (large and small)
  7. Pentane
  8. Glass Beads
  9. Sodium Sulphate (anhydrous)
  10. GC Vials
  11. 5mL Pasteur Pipets and Bulbs

Procedures:

Part 1: Preparation for Extraction

  1. Make 5 mL overnight cultures of yeasts to be sampled (18-20 hours @ 30 degrees). The media used here must be SD-Leu (glucose).
  2. Measure OD 600 of the cultures using the spectrophotometer. The OD 600 should be around 2.
  3. Add the remaining cultures to Erlenmeyer flasks and dilute with 50 mL of of SD-Leu.
  4. Grow up the cultures for about 2-3 hours (until OD 600 is 0.6-0.8).
  5. Transfer all of the cultures into falcon tubes and spin down the cells (5 min @ 2500g).
  6. Pour out media and resuspend the yeast pellets with about 47mL of SD-Leu (GPD) or SG-Leu (GAL).
  7. Transfer the cultures to Erlenmeyer flasks and grow up overnight (18-20 hours@ 30 degrees).
  8. Harvest cells (step 5).
  9. Weigh the large glass tubes and record values.
  10. Keep 1 mL of the media and transfer to large glass tubes and discard the rest of the media.
  11. Wash the yeast pellet with 5 mL of sdH2O.
  12. Transfer the 5mL of yeast to the glass tubes.
  13. Spin down the cells (5 min @ 2500 x g).
  14. Pour out water and weigh the glass tubes again to obtain the weight of the yeast pellet.
  15. Proceed to Extraction.

Part 2: Extraction (all of the following steps should be done in the fumehood in Bohlmann lab)

  1. Pipet in 2 mL of pentane to each glass tube.
  2. Add 0.5-1 mL of glass beads to each tube.
  3. Vortex as fast as possible without spilling for about 20 seconds.
  4. Pour solvent into smaller glass tube. Pipet the solvent out for the media samples using the pasteur pipets. Avoid glass beads!
  5. Add about 200 μL of Sodium Sulphate to the small glass tubes.
  6. Make one blank (4 mL of pentane and Sodium Sulphate).
  7. Repeat 1-4.
  8. Bring down the volume to about 1 mL by applying a stream of nitrogen.
  9. Place GC vials in a rack and label them.
  10. Pipet out solvent and transfer to vials.
  11. Apply nitrogen to vials and bring down the volume to about 0.4 mL.
  12. Add pentane to 0.5 mL line.

GC-MS Sample Preparation Protocol for bacteria

Supplies Needed:

  1. Erlenmeyer Flasks
  2. Appropriate Media [LB Broth and Terrific Broth)
  3. Spectrophotometer
  4. sdH2O
  5. Falcon Tubes
  6. GLASS test tubes (large and small)
  7. GC Vials
  8. 5mL Pasteur Pipets and Bulbs
  9. His-tag protein purification column
  10. A chem stand with a clamp
  11. Large centrifuge tubes
  12. Nanodrop

Reagents Needed:

  1. Lysis Buffer
  2. Wash Buffer
  3. Elution Buffer
  4. 0.5M NaOH
  5. GPP (geranyl pyrophosphate)
  6. Enzyme Assay Buffer
  7. Pentane
  8. DTT (final conc of 5 mM)
  9. Protein Inhibitory Cocktail (PIC)

Procedures:

Part 1: Preparation for Extraction

  1. Make 50 mL overnight cultures of selected colony to be sampled (18-20 hours @ 37 degrees). The media used here must be LB broth with the selectable antibiotics.
  2. Transfer 1 mL of overnight culture (in LB broth) to 250 mL of Terrific broth with the selectable antibiotics in a 1 L or 2 L Erlenmeyer flask.
  3. Grow up the cultures for about 4-5 hours @ 37 degrees (until OD600 is 0.8).
  4. Induce cells with 250 μL of 1M IPTG. Incubate culture for 16-18 hours at 16 degrees, shaking at 205 rpm.
  5. Pour the culture into the centrifuge tubes.
  6. Spin cells at 3000 rpm at 4 degrees for 20 minutes.
  7. Transfer the cultures to Erlenmeyer flasks and grow up overnight (18-20 hours @ 30 degrees).
  8. Carefully pour out supernatent and keep the pellet
  9. Proceed to extraction or keep pellet at -80 degrees

Part 2: Extraction

  1. Set up stand and clamp with the his-tag purification column. (Note: His-tag Column must never run dry. Therefore, it is advantageous to keep the column wet with wash buffer)
  2. Add 1 tablet of protease inhibitor cocktail and DTT (final conc of 5 mM) to 50 mL of Lysis Buffer
  3. Weigh out 1-1.5 g of cell pellet into falcon tube
  4. Add 5 mL of Lysis Buffer (containing protease inhibitor and TPP) to the cell pellet. Pipet up and down until the pellet is dissolved and the cell lysate is homogeneous.
  5. Sonicate cells (ready when there's a change in color; usually a lighter colour than the cell pellet)
  6. Centrifuge sonicated cells at max speed for > 20 min (until all cells are pelleted)
  7. Collect supernatent (should be around 5 mL).
  8. Transfer supernatent into the his-tag column. Adjust flow such that the drop of supernatent passing the column is about 1-2 drops per second. Supernatent can be passed through column twice.
  9. Add 10 mL of wash buffer. Collect drops slightly after the start of wash buffer flow through the column. Nanodrop to find out the absorbance and concentration of proteins passing through the column. Once this 10 mL of wash buffer has passed through, add another 10 mL of wash buffer to wash the column again to collect any residue proteins not binding to the beads of the column. Take a nanodrop reading at the end of the washing steps (last few drops of the wash buffer). Nanodrop reading should read 0 mg/mL at the end of second wash. If proteins are still present, continue with another wash until no protein can be found in the wash buffer.
  10. Elute with 5mL of elution buffer and collect into a falcon tube.

Part 3: GPP assay

  1. Pipet in 1 mL of purified protein to 4mL of enzyme assay buffer in a test tube
  2. Add 7 μL of GPP
  3. Add 1-2 mL of pentane (less is better in terms of getting high concentration yield; but may need large volume of pentane to avoid emulsion formation)
  4. Incubate in water bath for 1 hour at 30 degrees celsius
  5. Place GC vial in a rack and label it.
  6. Pipet out solvent (the less dense pentane layer) and transfer to vials to 0.5 mL line.
  7. Prepare a control without GPP added
  8. Freeze both samples
  9. Send for GC-MS