Spot Assay for Yeast

Materials and Reagents

1. Yeast cells
   
2. YES medium
   

Equipment

1. Multichannel Pipetman (Eppendorf)
   
2. OmniTray (V&P Scientific)
   
3. Microfuge tube
   
4. Standard laboratory spectrophotometer
   

Procedure

1. Start cultures from a 2 day old plate. Use pipette tip to pick up strains and resuspend them in 1.5 ml YES medium or water.
   
2. Vortex and transfer 1 ml to another microfuge tube. Test OD₆₀₀ for an accurate reading, the OD should be between 0.1 and 0.5.
   
3. Dilute the rest of the suspension to 16 OD₆₀₀, around 1.6 x 10⁶ cells per ml, 1,600 cells per μl. That is 4,800 or 5,000 cells per 3 μl. Spot 3 μl cells on each position.
   
4. If using OmniTray, start with the 1st column (8 wells in each column).
   
5. Do 5 fold serial dilution from the 1st to 5th column. Leave the 6th column empty. Transfer another 8 strains culture into the 7th column and do another 5 fold dilution.
   
   
   

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

1. Tong, Z., Gao, X. D., Howell, A. S., Bose, I., Lew, D. J. and Bi, E. (2007). Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition. J Cell Biol 179(7): 1375-1384.