UW-Stout/Bud Scars FA22
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
