Embryo Extract Solution (EE)

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Embryo Extract Solution (EE)
(makes 100 ml)

60 % L15
10 % FBS (Fetal Bovine Serum)
30 % ddH2O
1 % FB (Fungibact; Antibiotic antimycotic)

1. Remove jelly coat and membrane from embryos (stage 23-30) and count (you will need the number of embryos later).
2. Place embryos in tissue grinder, drawing off as much liquid as possible.
3. Add ~1 squirt of EE media.
4. Homogenize embryos (~ 50 plunges). Push down slowly and pull up quickly.
5. Transfer to 2 centrifuge tube and increase volume to ~ 30 mls using EE media or about 1/2 full. Balance both tubes.
6. Spin at 15K, 4C for 90 mins in SS-34 rotor.
7. Three layers appear. Bottom = Crude, Middle = Liquid, Top= Milky lipid layer. Carefully pipette middle layer. Avoid the top layer because it clogs the filter.
8. Add EE media such that the final concentration is 1 ml / 1 embryo.
9. Sterile filter.

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