Difference between revisions of "UW-Stout/Co Culture SP21"

From SGD-Wiki
Jump to: navigation, search
(Protocol)
(Protocol)
Line 18: Line 18:
 
# 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.  
 
# 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.
 
# 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.
+
  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.
 
#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.
 
#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

Equipment

Protocol

  1. Grow both the wild type and all knockout type yeast separate in double strength growth media cultures.
  2. 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.
  3. 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.
  4. 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.
  1. Incubate the plate within the incubator shaker for 48 hours at 30 degrees Celsius, shaking at 600 rpm.
  2. Take a 5 micro liter sample and mix it with 500 micro liters of saline to measure on the flow cytometer.