Difference between revisions of "UW-Stout/Salt Concentration SP21"
(Created page with "==Introduction== This protocol will be dependent on results of the highest salt concentration the yeast could grow in without much of a detrimental effect on it. Well 0 will...") |
|||
| Line 1: | Line 1: | ||
| − | ==Introduction== | + | ==Calibration Protocol== |
| + | |||
| + | ===Introduction=== | ||
This protocol will be dependent on results of the highest salt concentration the yeast could grow in without much of a detrimental effect on it. Well 0 will have the wild-type yeast strain and the following wells will have the knock-out strains. All samples will be treated with the same concentration results of the preceding protocol. Which was determined to be 700 mM of NaCl. | This protocol will be dependent on results of the highest salt concentration the yeast could grow in without much of a detrimental effect on it. Well 0 will have the wild-type yeast strain and the following wells will have the knock-out strains. All samples will be treated with the same concentration results of the preceding protocol. Which was determined to be 700 mM of NaCl. | ||
| − | ==Safety== | + | ===Safety=== |
For this experiment, baseline lab safety requirements are all that’s needed. Eye protection, lab coat, and gloves. | For this experiment, baseline lab safety requirements are all that’s needed. Eye protection, lab coat, and gloves. | ||
| − | ==Materials & Equipment== | + | ===Materials & Equipment=== |
* Salt (NaCl) | * Salt (NaCl) | ||
| Line 13: | Line 15: | ||
* Pipettes | * Pipettes | ||
* Pipette tips | * Pipette tips | ||
| − | * Yeast | + | * Yeast Strain (Wild Strain) |
* Media | * Media | ||
* Tubes | * Tubes | ||
| Line 22: | Line 24: | ||
* Basic lab safety equipment (listed in safety) | * Basic lab safety equipment (listed in safety) | ||
| − | ==Protocol== | + | ===Protocol=== |
# Vortex the yeast cultures to briefly to resuspend the yeast cells. | # Vortex the yeast cultures to briefly to resuspend the yeast cells. | ||
| Line 28: | Line 30: | ||
## Start off by creating a 2M salt solution by adding salt to media. (NaCl = 58.44 g/mol) | ## Start off by creating a 2M salt solution by adding salt to media. (NaCl = 58.44 g/mol) | ||
## Create 6 more diluted solutions following the values in the NaCl Conc. row listed in the chart below. | ## Create 6 more diluted solutions following the values in the NaCl Conc. row listed in the chart below. | ||
| − | # Set up 8 wells according to the table below. | + | # Set up 8 wells according to the table [[Stock Dilution Calculations|below.]] |
## To gain a better range this experiment will utilized 24 wells. This is done in order to replicate the experiment three times, expanding the data we would collect. | ## To gain a better range this experiment will utilized 24 wells. This is done in order to replicate the experiment three times, expanding the data we would collect. | ||
| − | ## In each well there will be 50 ul of yeast and media placed prior to adding the 50 ul of different salt concentrations to the wells. Well 0 will host the controlled variable (wild yeast strain with no concentration of salt added) but will add 50 ul of sterile water to that well instead. All wells will have a total volume of 100 ul. | + | ## In each well there will be 50 ul of the wild strain yeast and media placed prior to adding the 50 ul of different salt concentrations to the wells. Well 0 will host the controlled variable (wild yeast strain with no concentration of salt added) but will add 50 ul of sterile water to that well instead. All wells will have a total volume of 100 ul. |
| + | *Reference Stock Dilution Calculations listed below | ||
# Set up the plate reader as follows: | # Set up the plate reader as follows: | ||
## Temperature: 30°C | ## Temperature: 30°C | ||
| Line 42: | Line 45: | ||
| − | ===Stock Dilution Calculations=== | + | ====Stock Dilution Calculations==== |
To keep the added salt concentrations from being diluted in the sterile water, you will be doubling the mM concentration in the calculations to offset that foreseen issue. | To keep the added salt concentrations from being diluted in the sterile water, you will be doubling the mM concentration in the calculations to offset that foreseen issue. | ||
| Line 64: | Line 67: | ||
***Take 500 uL of 200 mM solution and place into new tube. Add 500 uL of sterile water to tube, vortex, and then label. | ***Take 500 uL of 200 mM solution and place into new tube. Add 500 uL of sterile water to tube, vortex, and then label. | ||
Add 50 uL of each concentration to its respective well location and move onto step 4. | Add 50 uL of each concentration to its respective well location and move onto step 4. | ||
| + | |||
| + | |||
| + | ==Final Protocol== | ||
| + | |||
| + | ===Introduction=== | ||
| + | This protocol will be dependent on results of the highest salt concentration the yeast could grow in without much of a detrimental effect on it. Well 0 will have the wild-type yeast strain and the following wells will have the knock-out strains. All samples will be treated with the same concentration results of the preceding protocol. This protocol would be replicated three times to produce a larger data set, but do to well availability and time constraints, we will be performing this experiment only once. | ||
| + | |||
| + | ===Safety=== | ||
| + | For this experiment, baseline lab safety requirements are all that’s needed. Eye protection, lab coat, and gloves. | ||
| + | |||
| + | ===Materials & Equipment=== | ||
| + | |||
| + | * Salt (NaCl) | ||
| + | * 96-well plate (need 36) | ||
| + | * Pipettes | ||
| + | * Pipette tips | ||
| + | * Yeast Strain (Wild Strain & Knock Out Strains) | ||
| + | * Media | ||
| + | * Tubes | ||
| + | * Sterile Water | ||
| + | * Vortex Machine | ||
| + | * Spectrophotometer | ||
| + | * Incubator set to 30°C | ||
| + | * Basic lab safety equipment (listed in safety) | ||
Revision as of 09:10, 27 April 2021
Contents
Calibration Protocol
Introduction
This protocol will be dependent on results of the highest salt concentration the yeast could grow in without much of a detrimental effect on it. Well 0 will have the wild-type yeast strain and the following wells will have the knock-out strains. All samples will be treated with the same concentration results of the preceding protocol. Which was determined to be 700 mM of NaCl.
Safety
For this experiment, baseline lab safety requirements are all that’s needed. Eye protection, lab coat, and gloves.
Materials & Equipment
- Salt (NaCl)
- 96-well plate (need 36)
- Pipettes
- Pipette tips
- Yeast Strain (Wild Strain)
- Media
- Tubes
- Sterile Water
- Vortex Machine
- Spectrophotometer
- Incubator set to 30°C
- Basic lab safety equipment (listed in safety)
Protocol
- Vortex the yeast cultures to briefly to resuspend the yeast cells.
- Create 7 different salt solutions. (Calculations listed in section below.)
- Start off by creating a 2M salt solution by adding salt to media. (NaCl = 58.44 g/mol)
- Create 6 more diluted solutions following the values in the NaCl Conc. row listed in the chart below.
- Set up 8 wells according to the table below.
- To gain a better range this experiment will utilized 24 wells. This is done in order to replicate the experiment three times, expanding the data we would collect.
- In each well there will be 50 ul of the wild strain yeast and media placed prior to adding the 50 ul of different salt concentrations to the wells. Well 0 will host the controlled variable (wild yeast strain with no concentration of salt added) but will add 50 ul of sterile water to that well instead. All wells will have a total volume of 100 ul.
- Reference Stock Dilution Calculations listed below
- Set up the plate reader as follows:
- Temperature: 30°C
- Mode: Kinetic
- Wavelength: 600 nm
- Interval: 10 minutes
- Total run time: 12 hours
- Transfer the assay plate to the reader and read for 12 hours.
Stock Dilution Calculations
To keep the added salt concentrations from being diluted in the sterile water, you will be doubling the mM concentration in the calculations to offset that foreseen issue.
- 2000 mM (2M) Stock Solution (Well 7)
- Add 116.88 mg NaCl into 1.5 mL centrifuge tube.
- Add media to the 1.0 mL mark on the tube
- Vortex and then label tube, 2 M
- Calculations for dilutions. Each new mM concentration will take stock from the previously diluted solution, starting from the 2 M master solution and working my way down.
- 1500 mM (Well 6)
- Take 750 uL of 2 M stock solution and place into new tube. Add 250 uL of sterile water to tube, vortex, and then label.
- 1000 mM (Well 5)
- Take 666.7 uL of 1500 mM solution and place into new tube. Add 333.3 uL of sterile water to tube, vortex, and then label.
- 700 mM (Well 4)
- Take 700 uL of 1000 mM solution and place into new tube. Add 300 uL of sterile water to tube, vortex, and then label.
- 400 mM (Well 3)
- Take 571.4 uL of 700 mM solution and place into new tube. Add 428.6 uL of sterile water to tube, vortex, and then label.
- 200 mM (Well 2)
- Take 500 uL of 400 mM solution and place into new tube. Add 500 uL of sterile water to tube, vortex, and then label.
- 100 mM (Well 1)
- Take 500 uL of 200 mM solution and place into new tube. Add 500 uL of sterile water to tube, vortex, and then label.
- 1500 mM (Well 6)
Add 50 uL of each concentration to its respective well location and move onto step 4.
Final Protocol
Introduction
This protocol will be dependent on results of the highest salt concentration the yeast could grow in without much of a detrimental effect on it. Well 0 will have the wild-type yeast strain and the following wells will have the knock-out strains. All samples will be treated with the same concentration results of the preceding protocol. This protocol would be replicated three times to produce a larger data set, but do to well availability and time constraints, we will be performing this experiment only once.
Safety
For this experiment, baseline lab safety requirements are all that’s needed. Eye protection, lab coat, and gloves.
Materials & Equipment
- Salt (NaCl)
- 96-well plate (need 36)
- Pipettes
- Pipette tips
- Yeast Strain (Wild Strain & Knock Out Strains)
- Media
- Tubes
- Sterile Water
- Vortex Machine
- Spectrophotometer
- Incubator set to 30°C
- Basic lab safety equipment (listed in safety)
