UV mutagenesis


• 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.