Home

About

Advanced Search

Browse by Discipline

Scientific Societies

E-print Alerts

Add E-prints

E-print Network
FAQHELPSITE MAPCONTACT US


  Advanced Search  

 
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
 

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 l 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 l 50 l 100 l 200 l
YPD added to wells 1 and 7 190 l 150 l 100 l 0 l
At the end of the experiment, when <10 CFU are observed in wells 1 and/or 7, spread culture (50-
100 l) directly on a YPD plate. The life span endpoint is defined as 0 CFU in 100 l (0.1 ml).
3. Use multichannel pipetter set to 50 l to mix contents of wells 1. Repipette slowly and evenly ~10
times to mix completely. Transfer 50 l 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 l per well, but this has a minimal effect on the
volume transferred to the YPD plate. This will yield the dilutions shown below:

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine