Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer
DEŞİFRE METNİ
– Allow Drosophila için lay eggs overnight onto a dish containing agar supplemented with grape juice için visualize eggs and coated with yeast için encourage flies için mate and lay eggs. To remove the eggs, add distilled water için the dish and gently wipe the surface with a paintbrush. Then pour the mixture through a nylon mesh placed videodan a plastic bushing için collect the eggs. Soak the eggs videodan two rounds of 7% bleach için remove the chorion, or protective outer layer, from the embryo. This process is called dechorionation.
The eggs are now stuck için the side of the bushing. Wash them three times with water için remove the bleach. Finally, use a paintbrush için transfer the eggs from the bushing için vials için continue development or for immediate use videodan an experiment. In the example protocol, we will collect and dechorionate Drosophila eggs under sterile conditions için rear flies with defined microbiota.
– Place nylon mesh into a plastic bushing için prepare the sieve for egg collection. If the same flies will be used the next day, immediately transfer them için a new cage containing freshly yeasted grape juice agar plates. The grape juice plate videodan the previous cage contains the fruit fly eggs. Remove dead flies with a clean paintbrush, and take caution not için break up the agar. Rinse the agar plate with distilled water and gently brush the eggs from the agar’s surface then pour the slurry over the mesh için collect the eggs.
Sterilize the biosafety cabinet with 70% ethanol then turn on the UV light. Spray all non-biological supplies with 70% ethanol and immediately place them videodan the biosafety cabinet, then sterilize them with UV light.
– It is critical için ensure that good aseptic technique is practiced when working within or when adding or removing new materials için the biosafety cabinet.
– Transfer the bushing with the eggs into a 120-milliliter specimen cup and slowly pour 90 milliliters of 0.6% sodium hypochlorite solution, or 7% bleach, into the bushing, just below the rim. Using forceps, periodically move the bushing up and down videodan the solution için resuspend the eggs. Transfer the bushing directly into a second specimen cup pre-filled with 90 milliliters of bleach.
At the end of the second bleach treatment, the eggs should begin için adhere için the sides of the bushing. Discard the bleach and completely rinse the bushing with sterile water. Again, use forceps için resuspend the egg several times during each wash. By the end of the third wash, most eggs should be attached için the side of the bushing.
Using a sterile paintbrush, transfer the eggs from the side of the bushing için the sterile diet. Leave the caps loose için allow oxygen için enter the tube. The transferred eggs will be visible on the surface of the diet.
