McClean: Intro to yeast

Overview

This introductory exercise is designed to get you started working with yeast. You will also take some data that will be useful to you anytime you are working with yeast in the lab.

Streaking Yeast out from Glycerol Stocks

• Wipe down your bench area with 70% ethanol to give yourself a clean workspace.
• Based on the yMM number, determine the location of the construct you want to streak out. It will be located in the -80°C freezer. Know where you are looking before you open the freezer door, sitting around with the door open is a no-no.
• Take the tube back to your bench. Ideally, store it on dry ice while streaking or be really quick.
• Take a sterile applicator stick. Scrape off a portion of the top of the frozen glycerol stock and streak it onto the edge of your plate.
• Return the construct to the -80°C freezer as quickly as possible. DO NOT LET THE GLYCEROL STOCK THAW! REPEATED FREEZE THAW CYCLES WILL KILL THE STOCK! If you need to streak out multiple constructs take out only 1-2 from the freezer at a time or store them on dry ice while streaking.
• Use sterile toothpicks to finish streaking the clone all over the plate so that you isolate single colonies. In the image below, (1) corresponds to your streaks directly from the glycerol stock and (2),(3),(4) correspond to fresh toothpicks. Use a new toothpick for numbers (2),(3),(4) or you won't achieve enough dilution to get nice single colonies.

Comparing Optical Density and Klett and Cell Count

By the end of this exercise you will have a useful graph that will allow you to convert between OD_{600}, Klett and cell count. This chart might vary with the strain you are using, so you should redo this exercise when you switch strains.

• Begin with a 50ml saturated overnight culture of yeast.
• Start an excel sh

Protocol

1. Step 1
2. Step 2
3. Step 3

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

Contact

• Megan N McClean 14:01, 20 July 2011 (EDT)

or instead, discuss this protocol.