Tuesday, November 18, 2003
Description UV Mutagenesis Procedure 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 l in 990l water). Further dilute 1:10 for mutagenized cells and 1:20 for unmutagenized cells. 9) Plate 100 l 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. Recipes Supplies Tips (责任编辑:泉水) |