| | |
Summary: Chronological Life Span Method Serial Dilutions and Pin Stamps
Materials: Sterile 96-well plates. YPD liquid medium. YPD-G418 plates (~20 ml, cured on bench for
1 day to dry). Sterile tips. Sterile reservoir. Multichannel pipetter.
1. Use a 96-well plate for dilutions and pin stamping. Use 6 wells for each sample (e.g., 1A-6A).
Use rows A-H for different samples. Do dilutions across columns 1-6 and 7-12. Up to 16 samples
fit on 1 96-well plate, but the best results are obtained using rows B-G.
2. Add YPD and yeast culture to wells 1 and 7. Fill wells 2-6 and/or 8-12 with 200 µl YPD. Record
culture volumes in Excel spreadsheet. The volume of yeast culture will depend on the rate of aging
over time. When <10 CFU (colony forming units) are observed in wells 2 and 8, increase the
volume of culture added to wells 1 and 7. Here are some examples:
Week 1 2 3 4
Culture added to wells 1 and 7 10 µl 50 µl 100 µl 200 µl
YPD added to wells 1 and 7 190 µl 150 µl 100 µl 0 µl
At the end of the experiment, when <10 CFU are observed in wells 1 and/or 7, spread culture (50-
100 µl) directly on a YPD plate. The life span endpoint is defined as 0 CFU in 100 µl (0.1 ml).
3. Use multichannel pipetter set to 50 µl to mix contents of wells 1. Repipette slowly and evenly ~10
times to mix completely. Transfer 50 µl to wells in next column until reaching wells 6. Do not
change tips in between (to avoid mistakes during serial dilution). Repeat for wells 7-12 if needed.
The last column (6 and 12) will contain 250 µl per well, but this has a minimal effect on the
volume transferred to the YPD plate. This will yield the dilutions shown below:
|
