Team:British Columbia/Protocols/Gcms

From 2011.igem.org

(Difference between revisions)
Line 4: Line 4:
Supplies Needed:
Supplies Needed:
-
1. Erlenmeyer Flasks
+
# Erlenmeyer Flasks
-
2. Appropriate Media [Glucose (GPD) or Galactose (GAL)]
+
# Appropriate Media [Glucose (GPD) or Galactose (GAL)]
-
3. Spectrophotometer                                                                                                              
+
# Spectrophotometer        
-
4. sdH2O
+
# sdH2O
-
5. Falcon Tubes
+
# Falcon Tubes
-
6. GLASS test tubes (large and small)
+
# GLASS test tubes (large and small)
-
7. Pentane
+
# Pentane
-
8. Glass Beads
+
# Glass Beads
-
9. Sodium Sulphate (anhydrous)
+
# Sodium Sulphate (anhydrous)
-
10. GC Vials
+
# GC Vials
-
11. 5mL Pasteur Pipets and Bulbs
+
# 5mL Pasteur Pipets and Bulbs
Steps:
Steps:
Line 20: Line 20:
Part 1: Preparation for Extraction
Part 1: Preparation for Extraction
-
1. Make 5mL overnight cultures of yeasts to be sampled (18-20 hours@ 30 degrees). The media used here must be SD-Leu (glucose).
+
# Make 5mL overnight cultures of yeasts to be sampled (18-20 hours@ 30 degrees). The media used here must be SD-Leu (glucose).
-
2. Measure OD600 of the cultures using the spectrophotometer. The OD600 should be around 2.
+
# Measure OD600 of the cultures using the spectrophotometer. The OD600 should be around 2.
-
3. 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 50mL of of SD-Leu.
-
4. 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 OD600 is 0.6-0.8).
-
5. 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).
-
6. 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).
-
7. 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).
-
8. Harvest cells (step 5).
+
# Harvest cells (step 5).
-
9. Weigh the large glass tubes and record values.
+
# Weigh the large glass tubes and record values.
-
10. Keep 1mL of the media and transfer to large glass tubes and discard the rest of the media.
+
# Keep 1mL of the media and transfer to large glass tubes and discard the rest of the media.
-
11. Wash the yeast pellet with 5mL of sdH2O.
+
# Wash the yeast pellet with 5mL of sdH2O.
-
12. Transfer the 5mL of yeast to the glass tubes.
+
# Transfer the 5mL of yeast to the glass tubes.
-
13. Spin down the cells (5 min @ 2500g).
+
# Spin down the cells (5 min @ 2500g).
-
14. 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.
-
15. Proceed to Extraction.
+
# Proceed to Extraction.
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)
-
1. Pipet in 2mL of pentane to each glass tube.
+
# Pipet in 2mL of pentane to each glass tube.
-
2. Add 0.5-1mL of glass beads to each tube.
+
# Add 0.5-1mL of glass beads to each tube.
-
3. Vortex as fast as possible without spilling for about 20 seconds.
+
# 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!
+
# Pour solvent into smaller glass tube. Pipet the solvent out for the media samples using the pasteur pipets. Avoid glass beads!
-
5. Add about 200uL of Sodium Sulphate to the small glass tubes.
+
# Add about 200uL of Sodium Sulphate to the small glass tubes.
-
6. Make one blank (4mL of pentane and Sodium Sulphate).
+
# Make one blank (4mL of pentane and Sodium Sulphate).
-
7. Repeat 1-4.
+
# Repeat 1-4.
-
8. Bring down the volume to about 1mL by applying a stream of nitrogen.
+
# Bring down the volume to about 1mL by applying a stream of nitrogen.
-
9. Place GC vials in a rack and label them.
+
# Place GC vials in a rack and label them.
-
10. Pipet out solvent and transfer to vials.
+
# Pipet out solvent and transfer to vials.
-
11. Apply nitrogen to vials and bring down the volume to about 0.4mL.
+
# Apply nitrogen to vials and bring down the volume to about 0.4mL.
-
12. Add pentane to 0.5mL line.
+
# Add pentane to 0.5mL line.

Revision as of 19:57, 15 August 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

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

Steps:

Part 1: Preparation for Extraction

  1. Make 5mL overnight cultures of yeasts to be sampled (18-20 hours@ 30 degrees). The media used here must be SD-Leu (glucose).
  2. Measure OD600 of the cultures using the spectrophotometer. The OD600 should be around 2.
  3. Add the remaining cultures to Erlenmeyer flasks and dilute with 50mL of of SD-Leu.
  4. Grow up the cultures for about 2-3 hours (until OD600 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 1mL of the media and transfer to large glass tubes and discard the rest of the media.
  11. Wash the yeast pellet with 5mL of sdH2O.
  12. Transfer the 5mL of yeast to the glass tubes.
  13. Spin down the cells (5 min @ 2500g).
  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 2mL of pentane to each glass tube.
  2. Add 0.5-1mL 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 200uL of Sodium Sulphate to the small glass tubes.
  6. Make one blank (4mL of pentane and Sodium Sulphate).
  7. Repeat 1-4.
  8. Bring down the volume to about 1mL 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 0.4mL.
  12. Add pentane to 0.5mL line.
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