Intro to yeast-PDF

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

The idea is to take a saturated culture of yeast (if it is truly saturated, it will max out to Klett and similarly provide little useful data on the spectrophotometer) and dilute it over a range and measure these diluted cultures using OD, Klett, and a hemacytometer to count cell number. This will tell you both the linear range of your measurement techniques, as well as the conversion parameters between the three measurement modalities.

• Begin with a 50ml saturated overnight culture of yeast in YPD.
• Start an excel sheet or similar to keep track of your data.
• Dilute your culture over an appropriate range into cuvettes, falcon tubes, or klett tubes (it’s your preference). You MUST use Klett tubes in the klett and you MUST use cuvettes in the spectrophotometer so figure out your preferred method. You can of course pour between one and the other.
• Measure the OD₆₀₀, Klett, and cell count using the hemacytometer. Protocols

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.