Search

Spot Assay for Yeast   

Download PDF How to cite Favorites 3 Q&A Share your feedback Cited by

In this protocol

Abstract

This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies.

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.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Tong, Z. (2012). Spot Assay for Yeast. Bio-protocol 2(1): e16. DOI: 10.21769/BioProtoc.16.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Bhavna Veer
Pondicherry University, India
Hi
Please let me know the procedure to make YPDA plates with the compound. I`m actually unable to validate my results with Spot Assay. I`ve used the concentration with which I got some sensitive mutants in 96 well liquid system. In fact, I have used a little higher concentration as well in the solid media. But the yeast mutant strains which have shown sensitivity to the drug in 96 well liquid system did not show any sensitivity in the plate system. Treated plate was as good as the control plate after the incubation.
Please help me with this.
Thank you.
5/8/2014 11:57:26 PM Reply
How I can explin my reselt if I use 25 ,28 ,30 ,31 ,34 ,36 c ?
I need explin the reselt plaese?
12/4/2012 12:18:06 AM Reply
Zongtian Tong
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

Sorry, I am not sure what do you ask. The 96 plate picture is just for reference. You can use any plate you like.

12/22/2012 9:49:56 PM


What is the easiest way to determine if a colony is yeast? Is there a simple test kit that you know of?

Thank you
Chandra
3/21/2012 12:04:02 AM Reply
Zongtian Tong
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

You can determine if the colony is yeast or bacteria by looking at the color and shape of the colony. Bacteria colony is usually yellowish and the shape is smooth.

You can also use a pipette tip to touch the colony, drop it into water and transfer some to a microscope slide. Observe the colony under light microscope. Bacteria is much smaller than yeast. E.Coli is rod shape. Budding yeast has buds.

3/22/2012 12:54:46 PM


Muhammad Sameeullah
Institute of Plant Science and Resources

How to concentrate yeast culture from OD600=0.70 to 1.0? please suggest easiest way. thanks.

9/16/2013 6:08:17 PM