Team:British Columbia/Protocols/Gcms

From 2011.igem.org

Revision as of 07:52, 28 September 2011 by Joeho604 (Talk | contribs)

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


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.


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. Pentane
  8. GC Vials
  9. 5mL Pasteur Pipets and Bulbs
  10. His-tag protein purification column
  11. A chem stand with a clamp
  12. Large centrifuge tubes

Part 1: Preparation for Extraction

  1. Make 50mL 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 1mL of overnight culture (in LB broth) to 250mL of Terrific broth with the selectable antibiotics in a 1L or 2L Erlenmeyer flask.
  3. Grow up the cultures for about 4-5 hours @ 37 degrees (until OD600 is 0.8).
  4. Induce cells with 250uL 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
Retrieved from "http://2011.igem.org/Team:British_Columbia/Protocols/Gcms"