Experiments on the analysis of yeasts with subsequent spores

Summary

The meiotic products are released from the ascospores, ruptured by sonication, and spread directly on agar plates, and the spore colonies can be screened for the target genotypes by the photocopy plate method.

Operation method

basic program

Materials and Instruments

Yeast
2-Hydroxyethanol Ethanol Yeast Lysate
Ultrasonic generator Probe Centrifuge

Move

1. Prepare cells for dissection of tetrads. If spores are formed on a plate, pick up the spores with a toothpick and place them in a 50 ml flask containing 5 ml of water; if spores are formed in liquid medium, add 1 ml of culture solution to 5 ml of water. Then add 0.5 ml yeast lyase-20T solution and 10 μl 2-mercaptoethanol and incubate overnight at 30°C on a shaker with gentle shaking.2. Add 5 ml of 1.5% NP-40 solution and transfer the mixed suspension to a disposable 15 ml test tube in an ice bath for 15 min.3. Clean the ultrasound probe before ultrasonication. Wash with water, then rinse with ethanol, and insert the ultrasound probe into a test tube containing a mixed suspension and into the liquid as far as possible without touching the bottom or walls of the tube. The tube was sonicated at 50% to 70% full power for 30 s, placed in an ice bath for 2 min, and repeated twice.

4. Centrifuge the spores at 1,200 g for 10 min, aspirate or pour off the supernatant, and resuspend the precipitate with 5 ml of 1.5% NP-40, oscillate vigorously on a vortex mixer, and repeat 2 times.

5. sonicate as in step 3 (repeat) and examine the spores after the last sonication.6. Centrifuge the spores at 1,200 g for 10 min, aspirate or pour off the supernatant, then resuspend with 5 ml of water and mix with vigorous shaking on a vortex mixer and repeat once.

7. 10-fold dilution of the treated spore suspension was counted using a hemocyte counting plate and diluted with water until the spore concentration was ¹⁰³ spores/ml Each plate was coated with 100 μl of the spore dilution and then incubated at 30°C for 3 days.

8. Photocopy the plates and screen for the target spore colonies.

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