1. Anethesize female using benzocaine - put ~5ml 6% benzocaine (stock solution in 100% ethanol) into 1L water.
   - leave animal until it is limp and completely unresponsive.
   - place animal onto paper towel
2. Remove ovaries (both) and wash with Ca2+, Mg2+ free Ringers
3. Dissociate overnight at 16°C in 0.1% collagenase in 5mg/ml ovalbumin in Ca2+/Mg2+-free Ringers supplemented with 10mM NaPO4 .Use rocker. To dissociate a complete ovary, use 20m total volume.
4. Recover oocytes and wash twice in OR2 (which can be supplemented with 5% dialyzed calf serum for long term culture) Media should supplemented with antibiotics - either gentamycin (50µg/ml) or penicillin/streptomycin (from 100X stock).
   

   Modified Ringers Solution (MRS) 110mM NaCl 2mM KCl 1mM MgCl2 2mM CaCl2 2mM NaHCO3 5mM HEPES pH 7.8

   OR2 media: 82.5mM NaCl 2.5mM KCl 1mM CaCl2 1mM MgCl2 1mM Na2HPO4 5mM HEPES pH 7.8

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Oocyte maturation:

Make oocyte media 5µg/ml progesterone (progesterone made as 5mg/ml stock in 100% ethanol).

Animal pole pigment clearing should be apparent by 3 to 4 hours, but may take as long as 6 to 8 hours to become evidence.

Even in cases where animal pole spot does not appear, the oocytes can have entered M-phase, as judged by disappearance of nucleus, breakdown of keratin filaments.

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Oocyte enucleation

transfer oocytes into MRS

use a pair of blunt forceps, gently hold oocyte and puncture with a unblunted 26/27 gauge needle.

gently squeeze oocyte until germinal vesicle appears. you can use the needle to tease GV out. use only enucleated egg in which GV is removed untorn.

note:usually the GV remains fairly round throughout the enucleation process, but sometimes it is almost stringy.

I would not use such oocytes.

place enucleated oocytes into MRS - allow to heal for 30 to 60 minutes. even some very ugly looking oocytes will heal completely under these conditions!

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Preparation of insoluble and soluble fractions from oocytes

Eggs and embryos must be dejellied with 2% cysteine pH 8.0 / wash 3x with Ringers.

Place oocytes, eggs or embryos into microfuge tube & remove excess liquid.

Typical experiment uses 5 to 20 oocyte/embryos per gel lane.
wash once in MSB buffer

homogenize first is MSB using 1.5ml in a 1.7ml microfuge tube.
carefully pipette up and down with pasteur pipette. be careful not to splash stuff out of the microfuge tube-it is easy to do!. -

centrifuge in microfuge for 15 minutes (at 4°C).

aspirate supernatant from the top of the tube

take care to remove yolk from the top of the "pellet" microfuge tube

re-suspend pellet in 1.5ml XEX

centrifuge resuspended pellet for 15 minutes at 4°C.

solubilize recovered pellet in either SDS-page or IEF sample buffer.

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