Difference between revisions of "UW Stout/Sucrose Fermentation SP22"
| Line 19: | Line 19: | ||
* Centrifuge | * Centrifuge | ||
* Vortex | * Vortex | ||
| − | |||
==Pilot Experiment Procedure== | ==Pilot Experiment Procedure== | ||
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# Remove supernatant | # Remove supernatant | ||
# Add 1 ml of PBS buffer to yeast pellet | # Add 1 ml of PBS buffer to yeast pellet | ||
| − | # Vortex | + | # Vortex for 30 seconds |
====Make Fermentation Solution==== | ====Make Fermentation Solution==== | ||
| − | # Mix the following ingredients for each | + | # Mix the following ingredients for each solution *See table 1 |
# Vortex solution for 30 seconds | # Vortex solution for 30 seconds | ||
# Store in 30°C incubator | # Store in 30°C incubator | ||
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{| border="1" cellspacing="4" cellpadding="2" | {| border="1" cellspacing="4" cellpadding="2" | ||
| | | | ||
| − | | | + | |Solution 1 |
| − | | | + | |Solution 2 |
| − | | | + | |Solution 3 |
| − | | | + | |Solution 4 |
|- | |- | ||
|Yeast | |Yeast | ||
| Line 84: | Line 83: | ||
===Results=== | ===Results=== | ||
| − | We found through qualitative analysis that the 4% total sucrose in | + | We found through qualitative analysis that the 4% total sucrose in solution produced the most CO2 after 48 hours. |
==Experimental Procedure== | ==Experimental Procedure== | ||
| Line 93: | Line 92: | ||
# Remove supernatant | # Remove supernatant | ||
# Add 1 ml of PBS buffer to yeast pellet | # Add 1 ml of PBS buffer to yeast pellet | ||
| − | # Vortex | + | # Vortex for 30 seconds |
| + | |||
| + | ====Make Fermentation Solution==== | ||
| + | # Follow solution 1 in table 1 for each gene | ||
| + | # Vortex solution for 30 seconds | ||
| + | # Store in 30°C incubator | ||
| + | # Sample solution (for ethanol production) in 24 and 48 hours. | ||
Revision as of 11:28, 3 May 2022
Contents
Introduction
The purpose of this protocol is to test if knockout yeast strains undergo fermentation, and if they do how much ethanol is produced. This will tell us if the gene that was knocked out plays a role in fermentation.
Materials
- 1 ml Yeast Culture (Approximately 1 million cells)
- You may need to dilute with sterile H2O to approximate the 1 million cells. Keep total volume at 1 ml.
- PBS Buffer Solution
- Yeast Growth Solution
- 0.85g YNB, 2.5 Ammonium Sulfate, 0.32g CSM, 5ml 100x Leucine, 100x Histine, 100x Uracil, 435 ml DI H2O
- 40% Sucrose Solution
Equipment
- High Performance Laser Chromatography (HPLC)
- Incubator
- Set to 30°C
- Centrifuge
- Vortex
Pilot Experiment Procedure
Set up yeast
- Collect 1 ml of yeast solution (approximately 1 million cells)
- Centrifuge for 3 minutes at 1000x gravity
- Remove supernatant
- Add 1 ml of PBS buffer to yeast pellet
- Vortex for 30 seconds
Make Fermentation Solution
- Mix the following ingredients for each solution *See table 1
- Vortex solution for 30 seconds
- Store in 30°C incubator
- Sample solution (for ethanol production) in 24 and 48 hours.
Table 1
| Solution 1 | Solution 2 | Solution 3 | Solution 4 | |
| Yeast | 1ml | 1ml | 1ml | 1ml |
| Growth solution | 8ml | 8ml | 8ml | 8ml |
| 40% Sucrose Solution | 1ml | 0.5ml | 0.25ml | 0.125ml |
| H20 | 0ml | 0.5ml | 0.75ml | 0.875ml |
| Total Volume | 10ml | 10ml | 10ml | 10ml |
| Total sucrose concentration in solution | 4% | 2% | 1% | 0.5% |
Results
We found through qualitative analysis that the 4% total sucrose in solution produced the most CO2 after 48 hours.
Experimental Procedure
Set up yeast
- Collect 1 ml of yeast solution (approximately 1 million cells)
- Centrifuge for 3 minutes at 1000x gravity
- Remove supernatant
- Add 1 ml of PBS buffer to yeast pellet
- Vortex for 30 seconds
Make Fermentation Solution
- Follow solution 1 in table 1 for each gene
- Vortex solution for 30 seconds
- Store in 30°C incubator
- Sample solution (for ethanol production) in 24 and 48 hours.
