Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine
http://hg.wustl.edu/hdk_lab_manual/yeast/yeast1.html
Purpose:
Before working with any yeast strain, begin with a newly isolated colony to avoid the possibility of working with a contaminated culture. Some yeast strains are unstable (e. g., small YAC-bearing strains) and need to be repurified by streaking on an agar plate and then verifying the genetic content of the isolated colony before proceeding. In cases where the strain is unstable, plan to streak the cells onto the selective medium to retain the desired stock, (however, most strains can be streaked onto the complete medium, YPD).
Time required:
Less than 5 minutes to streak the cells; 2-3 days for the colonies to grow.
Procedure:
- The yeast strain can be streaked from another plate, a slant (4 ml vial with cells growing on the surface of the media, which is hardened on a slant to produce greater surface area for the yeast cells to grow), or from a frozen glycerol stock. Take the wide, rounded end of a sterile flat toothpick and pick up a slightly visible amount of yeast cells.
- To transfer the cells to a media plate, begin the streak at one edge of the plate. Press the side of the toothpick containing the cells to the agar plate's surface and quickly streak the toothpick back and forth across part of the plate's surface (see #1 in the diagram below). The streaks should lie near one another, but should not cross over previous streaks.
- Turn the toothpick over to the side that did not originally touch the cells, or use a new toothpick for the next streaks. These streaks should start by crossing over the last streak, then proceeding as before into new areas of the agar plate (#2 in the diagram below).
- Repeat in new territory on the agar plate ( #3 in the diagram). The cells will be distributed on the plate in decreasing concentration through the streaks, and no matter how many cells were on the toothpick to begin with, there should be an area on the streaked plate that will produce isolated colonies.
- Cover the plate, invert it and incubate at 30 degrees C for 2-3 days. The plate should appear to have solid streaks of cells as well as isolated colonies. Pick a yeast colony well isolated from the others to use in subsequent work.
Plate appearance after 2 to 3 days' growth.
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