| Contributor: |
The Laboratory of Timothy Mitchison at Harvard University
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| The quality of the eggs is essential for good CSF extracts. Always sacrifice quantity for quality when trying to make functional extracts. Discard any batches of eggs that have "puff balls" or activated eggs that constitute more than 10% of the eggs. Use laid eggs and collect eggs at about 16 to 17 hours after priming with Progesterone (found in Pregnant Mare Serum Gonadotropin (PMSG)). If you are trying to make extracts that will form spindles that are competent for anaphase chromosome separation, it is necessary to use only freshly squeezed eggs. Keep the eggs cool (16°C incubator) and only bring them to room temperature right before you are ready to prepare the extract. |
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A. Procedure preparation
1. Get all solutions ready and put the 13 x 51 mm tubes in a rack (see Hint #2).
2. Have the Gelatin Solution at 37°C.
3. Coat a 150 x 75 mm Petri dish with 100 μl of the Gelatin Solution. Swirl the gelatin in the dish and replace it with XB.
4. Bring the frogs to room temperature at the last minute.
B. Procedure
1, Collect the laid eggs. Keep them in separate batches if there are distinguishable differences in their quality.
2. Suspend the eggs in 500 ml MMR in a 600 ml beaker and allow them to settle. Aspirate the MMR from over the eggs. Repeat this washing process until all of the dirt and debris are removed from the eggs.
3. Inspect for and remove any bad eggs using a cut off and flame-polished Pasteur pipette (See Hint #3 and #4).
4. Remove as much MMR from the eggs as possible.
5. Dejelly the eggs by suspending them in Dejellying Solution until the eggs are well-packed (about 7 to 10 min). Remove all of the Dejellying Solution.
6. Wash the dejellied eggs 2 or 3 times with XB in the Gelatin-coated Petri dish and remove all of the XB (See Hint #5).
7. Wash the eggs 2 or 3 times in 150 ml CSF-XB and remove as much Buffer as possible.
8. Wash the eggs 2 times in 100 ml CSF-XB plus 100 μl LPC.
9. Transfer the eggs into Ultraclear 13 X 51 mm tubes containing 1 ml of CSF-XB with LPC at 10 μg/ml and Cytochalasin D at 100 μg/ml. Allow the eggs to drop into the tubes.
10. Aspirate all of the Buffer from the top of the eggs.
11. Place the ultra clear tubes into a 15 ml tube and centrifuge for 10 sec at medium speed (setting 4) in a clinical centrifuge.
12. Remove all of the Buffer from the tubes and layer 1 ml of Versilube F-50 oil to the eggs.
13. Centrifuge the tubes at 2,000 rpm for 30 sec and then at 2,500-3,000 rpm (full speed) for 15 sec in a clinical centrifuge.
14. Remove all the Buffer and Versilube from the eggs, getting them as dry as possible.
15. Crush the eggs by centrifuging them in a SW55 Ti rotor with the full brake on at 10,000 rpm (10,000 X g) for 15 min at 16°C (see Hint #6).
16. Place the tubes on ice.
17. Wipe the exterior of the tube with 95% Ethanol. Collect the extract with an 18-gauge and syringe needle by puncturing the side of the tube near the bottom of the cytoplasmic layer and gently drawing the cloudy cytoplasmic layer into the barrel (see Hint #7).
18. Estimate the volume of the extract and add 0.001 volume LPC, 0.001 volume Cytochalasin D, Energy Mix to 1X, 0.025 volume of 2 M sucrose. The cytostatic factor (CSF) extract is now ready for use (see Protocol on Spindle Assembly in vitro).
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| LPC |
| 10 mg/ml Chymostatin
10 mg/ml Pepstatin
10 mg/ml Leupeptin
Make up in DMSO (CAUTION! see Hint #1)
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| Gelatin Solution |
| Warm to 37°C before use.
5% (w/v) Gelatin
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| Energy Mix (20X) |
| 150 mM Creatine Phosphate
20 mM ATP
20 mM MgCl2
Store in 100 μl aliquots at -20°C
2 mM EGTA
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| Dejellying Solution |
| 1X XB Salts
pH 7.8
2% (w/v) Cysteine
Make fresh within 1 hr of use
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| CSF-XB |
| 100 mM KCl
0.1 mM CaCl2
Make up fresh before use
10 mM HEPES, pH 7.7
50 mM Sucrose
5 mM EGTA
2 mM MgCl2
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| XB Salts (20X) |
| 2 mM CaCl2
2 M KCl
Store at 4°C
20 mM MgCl2
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| 95% (v/v) Ethanol |
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| XB |
| 100 mM KCl
0.1 mM CaCl2
Make up fresh before use
10 mM HEPES, pH 7.7
50 mM Sucrose
1 mM MgCl2
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| 10 mg/ml Cytochalasin D in DMSO (CAUTION! see Hint #1) |
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| MMR |
| 100 mM NaCl
Store at room temperature
2 mM CaCl2
2 mM KCl
1 mM MgCl2
5 mM HEPES, pH 7.8
0.1 mM EDTA
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| 0.5 M Potassium EGTA, pH 7.7 |
| Sterile filter and store at room temperature
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| 1 M HEPES, pH 7.7 |
| Sterile filter and store at 4°C
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| 2 M Sucrose |
| Sterile filter and store in aliquots at -20°C
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Versilube F-50 oil
Potassium EGTA
Cytochalasin D
HEPES
Gelatin
EDTA
Potassium Chloride
Sodium Chloride
Ethanol
ATP
EGTA
Sucrose
Calcium Chloride
Chymostatin
Magnesium Chloride
Pepstatin
Creatine Phosphate
Cysteine
Leupeptin
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Buffers and ambient temperature ideally should be at 18 to 20°C, and not above 23°C. Otherwise, a lower quality extract results.
3. "Bad" eggs, includes puffy, white eggs, lysed eggs, and activated eggs. Inspect for and remove bad eggs during the washes after dejellying as well, but do not take too much time to do so.
4. The flame-polished Pasteur pipette should have a diameter of 3 to 4 mm.
5. Do not pour the Buffer directly on the dejellied eggs. They are very fragile. For each wash, swirl the eggs around the dish and let the eggs settle back down. They should pack tight after the jelly coat is removed.
6. Ultracentrifugation at this step gives much more reproducible extracts
7. You should get about 0.5 to 0.75 ml of extract per tube. The cytoplasmic layer will be in the middle of the tube.
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Meth in Cell Biol 1999; 61:385-412
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