Yeast Life Span Assay



This protocol was submitted by Matt Kaeberlein.

Required Reagants:

Protocol:

  1. Streak yeast onto YPD plates and incubate overnight at 30C.
  2. Restreak onto fresh YPD the next day and incubate overnight at 30C.
  3. Patch onto fresh YPD early on 3rd day. Incubate at 30C for 2-3 hours. (I patch two strains per plate. Some people do one strain per plate. More than two is not recommended.)
  4. Dip end of micromanipulator into patch of cells. Move 50-100 cells about half way across plate from patch and deposit cells on agar.
  5. Align 40 to 50 cells per strain vertically on the plate in groups of 10 (see figure below).
  6. Incubate at 30C for 1.5-3 hours. Each of the cells you positioned in step 5 should divide one or two times.
  7. Using micromanipulator, obtain a virgin daughter cell (cells that have never produced a daughter cell) from each of the 40-50 original cells. Gather all remaining cells and deposit them in the graveyard (located near the original patch). This is the only time you will harvest daughter cells. After this always take the mother cell.
  8. Incubate for 2 hours at 30C.
  9. Separate daughter cells from each mother cell. Keep a tally of how many daughters are produced by each mother cell.
  10. Repeat steps 8 and 9 until all cells have stopped dividing. Place you plates at either 4C or 10C overnight. As the experiment progresses you will want to increase the incubation time in step 8 because the cell cycle is longer in older cells.
  11. Now you are ready to plot your data and find out whether your results are statistically significant