• Cells containing ADE3-URA3 plasmid grown in URA • dropout media
• YEPD plates
• sterile water
• petri dishes
• UV light source
• “light-proof” box
1) Grow cells 2-3 days to stationary phase
2) Wash cells 2x with water
3) Count cells (vortex well to separate cells as much as possible)
4) Resuspend to 2 x 108 cells/ml
5) Take 0.5 ml and resuspend in 15 ml water (~6.7 x 106 cells/ml). Set up 1 tube per mutagenesis condition
6) Pour cells into petri dish. Expose to UV light source for either 0, 1 or 2 min
7) Collect cells and place in dark
8) Dilute cells 1:100 (10 µl in 990µl water). Further dilute 1:10 for mutagenized cells and 1:20 for unmutagenized cells.
9) Plate 100 µl cells on YEPD plates (do this in the dark). This should yield approximately 300 live cells (hoping for 50% killing so plate approximately 600 total cells).
10) Place plates in box in 25¡ incubator until colonies are visible.
11) Count cells and calculate viability. Condition where 50% of cells are dead (ie: condition where mutagenized plate and unmutagenized plate have approximately the same number of colonies) is the condition that the scale up mutagenesis will be performed at.