Team:British Columbia/Protocols/Gcms

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 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. 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 5mM)
9. Protein Inhibitory Cocktail (PIC)

Procedures:

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

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 5mM) to 50mL of Lysis Buffer
3. Weigh out 1-1.5g of cell pellet into falcon tube
4. Add 5mL 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 > 20min (until all cells are pelleted)
7. Collect supernatent (should be around 5mL).
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 10mL 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 10mL of wash buffer has passed through, add another 10mL 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 1mL of purified protein to 4mL of enzyme assay buffer in a test tube
2. Add 7uL of GPP
3. Add 1-2mL 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
5. Place GC vial in a rack and label it.
6. Pipet out solvent (the less dense pentane layer) and transfer to vials to 0.5mL line.
7. Prepare a control without GPP added
8. Freeze both samples
9. Send for GC-MS