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    • CommentAuthorjuruble
    • CommentTimeJun 15th 2007 edited
     
    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.