Isolation of embryos and stage 14 oocytes

Preparing Stocks for Collection

Reagents:

Media bottles kept at 4°C

Dry yeast

Steps:

1. Sprinkle dry yeast into the media bottles.
2. Use CO₂ to anesthetize flies, count flies.
3. Stick the CO2 nozzle into the cotton of the bottle. Tip the bottle with the pipette tip on top of the bottle and tap on the bottom of the media bottle to cluster flies in one corner.
4. Normally add 80 flies/ bottle for WT flies
5. Count 80 flies create pile. Use the pile as an approximation of # of flies to add to each bottle.
6. After 2 days the flies have laid down embryos. You can either dispose of the adult flies in the ethanol fly morgue or you can flip the adults to a new bottle for more embryos.

Day 0:

Setting Up Cages for Embryo Collection

Reagents:

Apple Juice Agar Plates

Dry Yeast

Tap water

Equipement:

CO2 pad and nozzle

Clean Cages

Tape

Spatula

Plastic Beaker

Steps:

1. Make wet yeast in beaker: add dry yeast to 30 mL of water until solution become viscous and drips slowly
2. Rub about one finger full of wet yeast onto apple juice plates using a spatula. Make a circle.
3. Use CO₂ to anesthetize the flies in bottles.
4. Pour the flies into a cage. Place the cage with the mesh side down onto the CO2 pad.
5. Place the apple juice plate with yeast on top of the cage opening. Make sure there aren’t any openings for flies to escape. Tape the plate into place.
6. Leave the cage with apple juice plate on top. Once the flies wake up, you can turn the cage over with the juice plate on the bottom.
7. Next day, switch apple juice plates.
8. The day after (2 days after initial cage set up), transfer the flies into new cages. You will always want new cages for embryo collection.
9. Place new apple juice plates on top of the new cages.

Day 2:

Embryo Collection

Reagents:

Clean Cages

Apple Juice Agar Plates

Wet yeast

Bleach

Dry ice

Filter baskets 

Lysis Buffer A

DTT

Protease Inhibitor

Steps:

1. The day before collection, make apple juice/agar plates and leave in the incubator to warm overnight.
2. The next morning, switch the overnight plates for 2-hour plates.
3. During the 2 hours, make enough plates proportional to the number of cages and time points you plan on collecting. Also make spacer plates which will be reused.
4. Before spreading yeast on all the plates, use the 2-hour plate to gauge whether the flies prefer to lay embryos on the yeast or on the apple juice agar. If they prefer to lay on the apple juice agar, only spread a little bit of yeast on each plate to maximize the surface area of the apple juice agar. If they prefer to lay on the yeast, spread a broad sheet of yeast on each plate.
5. After 2 hours, switch the plate to a new plate to start embryo collection.
6. Leave the plate to collect for 1 hour.
7. Then switch the plate to a spacer plate.
8. Repeat steps 5 and 6 several times until you have all the time points you need.

Below is an example of a timeline if you started at 9 am and collected 5 time points.

1. Gather 1.5 ml eppendorf tubes to be used for embryo collection.
2. Prepare Lysis Buffer A, add Protease Inhibitor, DTT (No RNase Inhibitor). Turn on the centrifuge to cool to 4C. 
3. USE COLD BLEACH. Bleach will react with wet yeast to generate heat. If bleach isn’t cold, you’ll be cooking the embryos.
4.  Add cold bleach to 6-well plates. Leave for 5 minutes.
5. Meanwhile prepare, clean up, or flip last plate
6. Collect embryos by using a paint brush to place the embryos in a filter basket. Wash the embryos using 1X PBS.
7. Wash the embryos 3 times with 1X PBS. Use the 1X PBS to group the embryos to one corner of the filter basket. 
8. Transfer the filter basket to each well of the 6-well plates. Make sure all embryos are submerged under the bleach in the well. 
9. Gently shake the filter baskets in bleach for 2 minutes and 30 seconds. Immediately remove the embryos and thoroughly rinse them off with 1X PBS. 
10. Let the embryos dry. In the meantime collect next time point
11. Take a paint brush and gently collect the embryos and place them in an eppendorf tube containing roughly 50ul of supplemented lysis buffer A. Place the tubes on dry ice as you transport them back to the lab.
12. Once in lab, add 100- 150ul of lysis buffer A (supplemented). Grind embryos with dounce (like grinder) and place the tubes on regular ice for 5 minutes. Note: 50-100 embryos should have a total of 200 - 250ul of lysis buffer A. 
13.  Spin down the tubes and clarify tubes at 4°C 15,000 rpm for 5 min.
14. Transfer the supernatant to a new eppendorf tube. This is your lysate. Bradford for protein concentration.