Difference between revisions of "UW-Stout/Bud Scars FA22"
(Created page with " '''Materials''' *Wild type yeast culture *Knockout yeast culture *1.7ml microcentrifuge tube (1 for each culture) *Calcofluor White Stain Kit Reagent B- Calcofluor White (1....") |
|||
| Line 15: | Line 15: | ||
#Preserve cellular architecture and the composition of cells by adding 3.7% of formaldehyde and yeast cells to equal 1 ml of liquid | #Preserve cellular architecture and the composition of cells by adding 3.7% of formaldehyde and yeast cells to equal 1 ml of liquid | ||
#Vortex briefly and let sit for 50 minutes | #Vortex briefly and let sit for 50 minutes | ||
| − | #Centrifuge (13k for 60 seconds) to make a pellet and remove liquid without disrupting pellet | + | #Centrifuge (13k for 60 seconds) to make a pellet and remove liquid without disrupting the pellet |
| − | #Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), remove liquid without disrupting pellet | + | #Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), and remove liquid without disrupting the pellet |
#Repeat steps 4 and 5 | #Repeat steps 4 and 5 | ||
| − | #Pellet, remove liquid | + | #Pellet, remove liquid and suspend in 10 ul of calcofluor |
#Incubate at room temperature for 5 minutes | #Incubate at room temperature for 5 minutes | ||
#Centrifuge (13k for 60 seconds) remove liquid | #Centrifuge (13k for 60 seconds) remove liquid | ||
#Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), remove liquid | #Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), remove liquid | ||
#Resuspend pellet in 50 ul of water | #Resuspend pellet in 50 ul of water | ||
| − | #Mount on slide (5ul) and cover with coverslip using petroleum jelly around the edges of the coverslip to prevent loss of moisture | + | #Mount on the slide (5ul) and cover with a coverslip using petroleum jelly around the edges of the coverslip to prevent loss of moisture |
#Put a paper towel below and above and cover with a heavy object for 10 minutes | #Put a paper towel below and above and cover with a heavy object for 10 minutes | ||
| − | #Look at the cells under a microscope and observe the | + | #Look at the cells under a microscope and observe the number of bud scars on each cell. View 100 different cells |
Revision as of 15:16, 20 December 2022
Materials
- Wild type yeast culture
- Knockout yeast culture
- 1.7ml microcentrifuge tube (1 for each culture)
- Calcofluor White Stain Kit Reagent B- Calcofluor White (1.0g), Evans Blue Dye (0.4g), and demineralized Water (1000.0ml) (10 ul for each culture)
- Glass Microscope Slide
- Coverslip
- Microscope
- Distilled Water ()
Procedure
- Gather materials
- Preserve cellular architecture and the composition of cells by adding 3.7% of formaldehyde and yeast cells to equal 1 ml of liquid
- Vortex briefly and let sit for 50 minutes
- Centrifuge (13k for 60 seconds) to make a pellet and remove liquid without disrupting the pellet
- Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), and remove liquid without disrupting the pellet
- Repeat steps 4 and 5
- Pellet, remove liquid and suspend in 10 ul of calcofluor
- Incubate at room temperature for 5 minutes
- Centrifuge (13k for 60 seconds) remove liquid
- Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), remove liquid
- Resuspend pellet in 50 ul of water
- Mount on the slide (5ul) and cover with a coverslip using petroleum jelly around the edges of the coverslip to prevent loss of moisture
- Put a paper towel below and above and cover with a heavy object for 10 minutes
- Look at the cells under a microscope and observe the number of bud scars on each cell. View 100 different cells
