High Speed Egg Extract Preparation

1. 20X Extract buffer (XB) salt stock: 2 M KCl, 20 mM MgCl2,2 mM CaCl2, filter-sterilize and store at 4°C.

2. Extract buffer (XB): 1X XB salts (100 mM KCl, 0.1 mM CaCl2,1 mM MgCl2; from 20X XB salts stock solution), 50 mM sucrose (1.5 M stock; filter-sterilize and store in aliquots at -20°C), 10mM HEPES (1 M stock, titrated with KOH such that pH is 7.7 when diluted to 10 mM; should require about 5.5 mL of 10 N KOH for 100 mL; filter-sterilize, and store in aliquots at -20°C) (see Note 4). Prepare about 100 mL.

3. 2% (w/v) Cysteine (Sigma C-7755): Made up in 1X XB salts within 1 h of use and titrated to pH 7.8 with NaOH. Prepare about 300 mL.

4. CSF-XB: 1X XB salts (100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2), 1 mM MgCl2 (in addition to MgCl2 present in XB salts; final concentration 2 mM), 10 mM potassium HEPES, pH 7.7, 50 mM sucrose, 5 mM EGTA, pH 7.7. Prepare 50 mL.

5. Protease inhibitors: Mixture of leupeptin (Boeringer Mannheim 1017 101), chymostatin (Boeringer Mannheim 1 004 638), and pepstatin (Boeringer Mannheim 600 160), each dissolved to a final concentration of 10 mg/mL in dimethyl sulfoxide (DMSO). Store in small aliquots at -20°C.

6. 1X MMR Prepare as described in Subheading 2.1., item 2.

7. 1 M CaCl2 (filter-sterilize and store at 4°C).

8. Versilube F-50: Made by General Electric. Can be purchased from Andpak-EMA (1560 Dobbin Drive, San Jose, CA 95133; Tel. [408]-272-8007).

9. Energy mix (store in aliquots at -20°C): 150 mM creatine phosphate (Boeringer Mannheim Biochemicals 127 574), 20 mM ATP (Boeringer Mannheim Biochemicals 519 979), 20 mM MgCl2, Store in 0.1-mL aliquots at -20°C.

10. Pregnant mare serum gonadotropin (PMSG).: 100 U/mL PMSG (367222; Calbiochem, San Diego, CA). Made up in water and stored at -20°C.

11. Human chorionic gonadotropin (HCG): 1000 U/mL HCG (Sigma CG-10 from human pregnancy urine). Made up in water and stored at 4°C.

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