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 Remove ovaries (both) and wash with Ca2+, Mg2+ free Ringers 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. 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 (50 g/ml) or penicillin/streptomycin (from 100X stock).Modified Ringers Solution (MRS) 110mM NaCl2mM KCl1mM MgCl2 2mM CaCl22mM NaHCO35mM HEPES pH 7.8 OR2 media:82.5mM NaCl2.5mM KCl1mM CaCl21mM MgCl21mM Na2HPO45mM HEPES pH 7.8 back to the topOocyte maturation:Make oocyte media 5 g/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.top of this page transfer oocytes into MRSuse 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!top of this page Preparation of insoluble and soluble fractions from oocytesEggs 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 bufferMedium salt extraction buffer150mM NaCl 50mM NaF10mM EDTA10mM Tris-base pH 7.4 XEX buffer: 1.5M KCl 0.3M sucrose 50mM NaF10mM EDTA.10mM Tris pH 7.4 0.5% NP40homogenize 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.