Difference between revisions of "UW-Stout/Co Culture SP21"
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==Materials== | ==Materials== | ||
* Double strength [https://sunrisescience.com/shop/growth-media/media-for-yeast/sd-complete-ynbnitrogen-glucose-csm-supplement-mix/sd-broth/sd-powder-10-x-0-5-liter-pouches/ synthetic-defined media] (liquid) | * Double strength [https://sunrisescience.com/shop/growth-media/media-for-yeast/sd-complete-ynbnitrogen-glucose-csm-supplement-mix/sd-broth/sd-powder-10-x-0-5-liter-pouches/ synthetic-defined media] (liquid) | ||
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==Equipment== | ==Equipment== | ||
| − | *[http://www.southwestscience.com/SBT1500H_Heated_Plate_Vortexer.html Southwest Science heated plate shaker set to 600 rpm and 30°C] | + | *[http://www.southwestscience.com/SBT1500H_Heated_Plate_Vortexer.html Southwest Science heated plate shaker set to 600 rpm and 30°C] with these [[Media:UWStoutSpring2021CoCultureKnockoutExperimentFlowcytometerSettings.txt | settings]] |
| − | *flow cytometer | + | *[https://www.luminexcorp.com/guava-easycyte-flow-cytometers/#overview 4th generation Guava® easyCyte™ flow cytometer] |
| − | *type 1000, 200, and 20 micro | + | *lab vortex mixer |
| + | *type 1000, 200, and 20 micro pipettes | ||
==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 flow cytometer 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 micro liters 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. |
| + | #The yeast will likely have clumped into pellets at the bottom of the growth medium after 48 hours. Pipette them up and down to mix them back into the growth medium. | ||
| + | #Take a 5 micro liter sample and mix it with 495 micro liters of saline to create a 1:100 dilution and use a vortex to ensure the yeast is fully re-suspended. Measure the composition of the sample on a flow cytometer. | ||
Latest revision as of 13:26, 27 April 2021
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 with these settings
- 4th generation Guava® easyCyte™ flow cytometer
- lab vortex mixer
- 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 flow cytometer 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 micro liters 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.
- The yeast will likely have clumped into pellets at the bottom of the growth medium after 48 hours. Pipette them up and down to mix them back into the growth medium.
- Take a 5 micro liter sample and mix it with 495 micro liters of saline to create a 1:100 dilution and use a vortex to ensure the yeast is fully re-suspended. Measure the composition of the sample on a flow cytometer.
