Difference between revisions of "UW-Stout/Co Culture SP21"
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==Protocol== | ==Protocol== | ||
| − | # Create concentration of 100 cells per micro liter for both the knockout and control fluorescent | + | # Grow both the wild type and all knockout type yeast separate in double strength growth media cultures. |
| − | # | + | # Use a flowcytometer to measure the cell concentration of a 1:100 dilution in saline of both the wild type and knockout cultures. The goal is to measure the culture stock concentration. |
| − | # | + | # Create a concentration of 100 cells per micro liter for both the knockout and control fluorescent strains from the cultures in single strength growth media. |
| − | # Incubate the plate within the incubator shaker for 48 hours at 30 degrees Celsius, shaking at 600 rpm. | + | # Using the concentrations from step 3, take 5 microliters of each strain and assign them to a well in the micro assay plate. Then, add 5 micro liters of the control strain into each of these well. Fill each well with 990 micro liters of growth media for a total of 1 mL each. |
| − | # | + | ## Note: If 100 cells per micro liter cannot be reached for a knockout type strain, match the control type to its max concentration for that specific well. The goal is to have equal cell concentration of each of the control and knockout strains in the well. |
| + | #Incubate the plate within the incubator shaker for 48 hours at 30 degrees Celsius, shaking at 600 rpm. | ||
| + | #Take a 5 micro liter sample and mix it with 500 micro liters of saline to measure on the flow cytometer. | ||
Revision as of 14:04, 13 April 2021
This page is under construction
Materials
- Double strength synthetic-defined media (liquid)
- Single-strength synthetic-defined media (liquid)
- Phosphate buffered saline, sterile.
- Glycerol stocks of knockout strains
- Thomas Scientific square,deep 96 well, round bottom micro assay plate
Equipment
- Southwest Science heated plate shaker set to 600 rpm and 30°C
- 4th generation Guava® easyCyte™ flow cytometer
- type 1000, 200, and 20 micro pipettes
Protocol
- Grow both the wild type and all knockout type yeast separate in double strength growth media cultures.
- Use a flowcytometer to measure the cell concentration of a 1:100 dilution in saline of both the wild type and knockout cultures. The goal is to measure the culture stock concentration.
- Create a concentration of 100 cells per micro liter for both the knockout and control fluorescent strains from the cultures in single strength growth media.
- Using the concentrations from step 3, take 5 microliters of each strain and assign them to a well in the micro assay plate. Then, add 5 micro liters of the control strain into each of these well. Fill each well with 990 micro liters of growth media for a total of 1 mL each.
- Note: If 100 cells per micro liter cannot be reached for a knockout type strain, match the control type to its max concentration for that specific well. The goal is to have equal cell concentration of each of the control and knockout strains in the well.
- Incubate the plate within the incubator shaker for 48 hours at 30 degrees Celsius, shaking at 600 rpm.
- Take a 5 micro liter sample and mix it with 500 micro liters of saline to measure on the flow cytometer.
