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 (3,050 ul)

Procedure

1. Gather materials
2. Preserve cellular architecture and the composition of cells by adding 3.7% of formaldehyde and yeast cells to equal 1 ml of liquid
3. Vortex briefly and let sit for 50 minutes
4. Centrifuge (13k for 60 seconds) to make a pellet and remove liquid without disrupting the pellet
5. Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), and remove liquid without disrupting the pellet
6. Repeat steps 4 and 5
7. Pellet, remove liquid and suspend in 10 ul of calcofluor
8. Incubate at room temperature for 5 minutes
9. Centrifuge (13k for 60 seconds) remove liquid
10. Add 1 ml of water, vortex briefly, centrifuge (13k for 60 seconds), remove liquid
11. Resuspend pellet in 50 ul of water
12. Mount on the slide (5ul) and cover with a coverslip using petroleum jelly around the edges of the coverslip to prevent loss of moisture
13. Put a paper towel below and above and cover with a heavy object for 10 minutes
14. Look at the cells under a microscope and observe the number of bud scars on each cell. View 100 different cells