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Fly Embryo Collection.md

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Fly Embryo Collection.md

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PREPARING THE AGAR PLATE

  1. Place flies in a collection cage with an apple juice agar plate. Give them at least a day to acclimate to the cage and start laying more eggs before doing a collection.
  2. When the flies are ready, spread yeast in the middle of a fresh apple juice agar plate, place the plate on the cage for 2 hours, then remove.
  3. Leave the plate at room temperature until it reaches the desired developmental stage. The embryos will continue to develop until they are placed in the heptane/PFA mixture -- plan your schedule accordingly.

REMOVING THE SHELLS

  1. Remove any remaining yeast from the plate.
  2. Place bleach on the agar plate for 1 minute, while swirling the plate or using a nylon bristle brush to loosen the embryos from the agar.
  3. In a quick movement, pour the embryos into a collection tube fitted with mesh under the gasket. Rinse with water for 1 minute.

FIXING THE EMBRYOS

  1. In the hood, unscrew the collection tube and dip the mesh embryo-side down into a scintillation vial filled with 5 mL of heptane and 5 mL of 8% PFA + 50 mM EGTA in 1x PBS (To make 40 mL: 10 mL 32x PFA + 4 mL .5M EGTA + 4 mL 10x PBS + 22 mL H20).
  2. Use a paintbrush to collect any embryos stuck to the mesh or inside of the collection tube.
  3. Place the scintillation vial on the shaker table for 25 min at 400 rpm.
  4. The PFA mixture will be on the bottom, the heptane will be on the top and the embryos should float between the 2 layers.

CLEAN UP AND STORAGE

  1. In the hood, pipette off the PFA mixture from the bottom of the vial and put in hazardous waste. Take care not to get embryos in the pipette tip -- they are sticky and difficult to retrieve.
  2. Once the PFA mixture is removed, add 5 mL of 100% methanol and shake/vortex for approximately 3 minutes. Most of the embryos should sink the bottom of the vial. IF they mostly float, add more methanol and continue vortexing until they sink.
  3. Tap down the vial on a slight angle so the embryos are collect together along one side of the bottom. Use a razor blade to cut 1/4" off the end of a 1000uL pipette tip at an angle, and use the angled tip to pipette the embryos into a 1.7 mL tube. Rinse the embryos 2x or 3x with fresh 100% methanol. Store in freezer in 1 mL of 100% methanol or use for cryo-sectioning. Place used heptane and methanol into flammable waste.