Experiments on the preparation and dissection of tetrads of Saccharomyces cerevisiae
Experiments on the preparation and dissection of tetrads of Saccharomyces cerevisiae
A standard light microscope is required for yeast tetrad analysis, and the microscope stage should be able to be moved along the x and y axes and the distance traveled can be accurately measured, but not moved up and down.
Operation method
basic program
Materials and Instruments
Yeast Move 1a. Glusulase treatment: Wash the spores with sterile water for 3 times, then resuspend with 5 ml of sterile water and take 30 μl of diluted in 270 μl of sterile water. Observe the cells under optical microscope, detect the intact ascospores, add 3 μl of diluted Glusulase enzyme to the spore preparation solution, gently shake with a vortex mixer, incubate at room temperature for 2 min, then observe the cells under microscope, and continue to incubate at room temperature until the Glusulase enzyme treatment is complete.1b. Yeast Lysin-100T treatment: Cells were resuspended with 50 μl of Yeast Lysin-100T solution. Observe the intact ascospores under a light microscope. incubate at 30°C for 10 min and slowly add 0.8 ml of sterile water along the wall of the test tube.2. Place the digestion tube in an ice bath and draw two parallel lines on the surface of the YPD dissection plate with an inoculating loop dipped in treated spores. Common Problems For more product details, please visit Aladdin Scientific website.
Yeast Lysin Sorbitol
Dissecting microscope Dissecting needle
3. Observe the inverted plate under a dissecting microscope to visualize individual tetrads, arranged in tetrahedral or diamond-shaped clusters of spores.4. Secure the plate so that the scribe line on the plate is parallel to the x-axis of the platform, and select an individual quadrant to focus and position in the center of the field of view. Adjust the position of the dissection needle upward using the coarse adjustment knob so that the needle contacts the surface of the plate when the joystick is pushed approximately halfway.
5. Gently touch the needle to the surface of the plate, close to and pick the quadrant.6. Move the plate, position the needle 1 cm from the line and perpendicular to the line of the tetrad spores, gently touch the needle to the surface of the agar and place the tetrad spores on the plate, the following may occur during the operation:
(1) No spores are laid down. In this case, repeat step 6, applying a little more force when the needle touches the agar.(2) With only 1 spore on the plate, move about 0.5 cm along the x-axis Repeat step 6.
(3) If there are 2 or 3 spores on the plate, then move about 1 cm along the y-axis and repeat step 6 to place 1 or more spores. Rotate the joystick to separate the spores so that the tip of the needle impinges on the plate and brings out the spores, picking out and repositioning the spores so that the spores are positioned perpendicular to the original scribe line and 0.5 cm apart from each other.
(4) If four spores are present, the tip of the needle should be used to separate them. Once the above has been accomplished, pick one or more spores and point them so that they are positioned perpendicular to the original scribe line and each 0.5 cm apart.7. Using the x- and y-axis control knobs, move the plate back so that the dissecting needle is directly under the line of treated spores. Repeat steps 5 and 6 to place each tetrad spore at regular intervals on a straight line, 1 cm from and parallel to the original tetrad line.
8. The position of these lines can be determined by drawing a marked line at random on the movable platform and aligning it with a fixed x-axis scale that is level with the back edge of the platform. Alternatively, a strip of paper marked at 0.5 cm intervals can be attached along the front edge of the platform, with the lower right corner of the plate holder used as the arbitrary alignment point.9. Continue repeating steps 5 to 8 until all positions along the x-axis are filled with dissected tetrapods, rotate the flat plate 180 degrees , and dissect the other tetrapods from the corresponding scribe lines on the other side of the flat dish. Each side of the flat plate can be dissected into 13 tetrads.
Figure 1. Dissecting microscope