Please find the following protocol:

Yeast Tetrad Dissection

Overview:

After two yeast strains are mated, the resulting diploid can be sporulated and the progeny analyzed. The four haploid spores produced from each meiosis are enclosed in an ascus. This is known as a tetrad. Following the digestion of the ascus wall, the spores of the tetrad can be separated and germinated. Thus, the segregation of any marked gene can be analyzed. This protocol describes the technique of dissecting tetrads.


Procedure:

A. Digestion of Sporulated Diploids

1. Check that the yeast diploid cells previously placed on sporulation media have sporulated by examining them under the microscope. To do this, take a small bit of cells off the sporulation plate with a sterile toothpick and mix with 10 μl of sterile ddH2O. Use the 40X objective to determine the percentage of 4-spore tetrads (Different yeast strains differ in their efficiency in sporulation. For W303, 40% to 50% sporulation is very good. Some strains have a sporulation rate of only 10%). Often the fourth (and third) spore of a tetrad will be above or below the plane of focus of the other spores in the ascus.

2. Place 10 μl of 5 mg/ml Zymolyase in a sterile microcentrifuge tube.

3. Scrape a small blob of sporulated cells with a sterile toothpick into the tube and mix.

4. Incubate for 8 to 10 min at room temperature.

5. Dilute the enzyme/spore mixture with 100 μl of sterile ddH2O.

6. Place the tube of diluted, digested spores on ice.

B. Preparation of Dissecting Plate

1. Chose a YPD Plate that was evenly poured and ideally a couple of days old. Mark a line down the middle of the plate (not the lid) with a black marker. Freshly made plates are too wet and are difficult to use because the tetrads don't behave well and stick to the needle. Plates that are a couple of days old still have a thin layer of moisture that creates the surface tension necessary to transfer spores from the needle to the agar.

2. Use a sterile 1 ml glass pipet or a sterile loop to dip into the digested tetrads and gently streak along one quarter of the line. Repeat three more times until the entire line has been covered.

3. Take the plate to a dissecting scope equipped with a micromanipulator. Set the plate on the microscope stage so that the marked line is horizontal.

4. Situate the place such that a position near the edge of the plate is centered under the lens. By eye, bring the needle to the center of the field and near the surface of the agar. Using 10X magnification, look for the needle within the field of vision. Use the joystick of the micromanipulator to move the needle to the center of the field and very close to the surface of the agar.

5. Change the lens to a higher magnification. Using the micromanipulator joystick, gently bring the needle down to touch the agar. You should see a dark ring and/or a change in refraction when the needle touches the agar (There is a slight film of water that forms between the needle and the agar). Focus on the surface of the agar.

6. Plunge the needle deeply into the agar several times to remove any spores that may have been left on the needle by previous users.

C. Dissection of Tetrads

1. Lift the needle so that it will not drag on the agar and move the plate so that the black line is visible.

2. Look for tetrads among the cells put down, refocusing slightly if needed. Tetrads appear as clusters of four spores in a diamond pattern. Avoid clusters of 4 spores or cells that do not have this diamond arrangement; they could simply be 4 digested spores that were deposited next to each other.

3. Chose a tetrad that is not sitting right next to other cells or spores to avoid picking up more than a single tetrad. It will be hard to distinguish the spores of the tetrad from the other spores and cells after the needle contents are set down.

4. Bring the needle down to pick up the chosen tetrad. This takes practice. There are several different ways to do this. One was is to "scoop" up the tetrad, changing both the X and Z axis controls (or both the Y and Z axis controls) to bring the needle down at an angle toward the tetrad. It is also possible to bring the needle straight down slightly to one side of the tetrad. Lightly touching the tetrad should cause the tetrad to stick to the tip of the needle, due to surface tension of moisture on the tip of the needle. Quickly bring the needle up after touching the plate. As the needle is pulled away from the plate, the film of water separates. The surface tension of the water on both the needle and the agar can pull the spores or tetrad toward either surface. Pulling the needle away quickly seems to facilitate the attraction of the tetrad for the needle.

5. If the tetrad no longer appears in the field of vision, assume that it is on the needle tip. Move the needle up so that is won't drag along the agar.

6. Move the plate to the position where you want to place the first spore of the tetrad. Usually spores of a tetrad are placed at least 10 cm from the line of digested tetrads and cells placed on the plate and laid down in a line 5 mm apart from each other. Bring down the needle, lightly touching the agar, to deposit the tetrad. It often helps to tap the microscope or the table just as the needle touches the agar or leaves the agar to facilitate dropping the tetrad or spores.

7. Sometimes the tetrad will break up right away. Other times it is necessary to repeatedly pick up and drop the tetrad to separate the spores. Leave one spore and pick up the others to move to their designated positions. Again, tapping the microscope or the table just as the needle touches the agar or leaves the agar will help release the spores from the needle.

8. Using the scale on the microscope stage, place each of the individual spores at designated places for that tetrad.

9. Be careful when depositing spores to a new location on the plate. The agar depth may have changed and it will be necessary to refocus on the new plane of the agar surface. It is not possible to see if the spores have been deposited onto the agar if the focus is not on the agar surface.

10. Twelve dissected tetrads will fit on one plate.

11. Place the YPD Plate at 30°C for several days, unless some of the segregants are temperature sensitive, in which case place the plate at the permissive temperature.

D. Troubleshooting

1. If it is not possible to focus on the cells on the agar surface because the objective pushes against the bottom of the plate as you attempt to focus, then chose a new YPD plate that is thinner. The agar of the original plate is thicker than the focal distance of the objective.

2. If a tetrad or spore seems to be "lost" on the needle or to have disappeared, go to an empty spot on the edge of the plate and sink the needle deeply into the agar several times to clean the tetrad off the needle. Cells will not fall off the needle on their own. They are either stuck to the needle or on the agar. If this problem happens repeatedly, check that the needle surface is perpendicular to the plate, is free of chips or nicks, and is clean of bits of agar.

3. If any extra spores suddenly appear or if you have any suspicion that not all of the spores on the needle are spores from the same tetrad, then record that information for that location on the plate, make sure the needle is clean by sinking the needle deeply into the agar several times, and start with a new tetrad.

4. Nothing seems to work. A variety of variables affect the ease of dissecting tetrads, including the moisture and evenness of the agar, the condition of the needle, the degree of digestion of the tetrads, and the ambient humidity.


Solutions:

5 mg/ml Zymolyase
Store at -20°C.

YPD Plates
20 g/liter Agar
10 g/liter Yeast Extract
Cool somewhat before pouring plates
20 g/liter Peptone
Autoclave for 30 min
20g/liter Glucose (Dextrose)


Bioreagents and Chemicals:

Yeast Extract
Peptone
Zymolyase
Agar
Glucose


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p9049