CSF extract, sara's protocol (Stukenberg lab) and Cycling extracts, mike's protocol (Stukenberg lab): Difference between pages

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Sara's CSF EXTRACT
   
   
Preparation:
Mike’s Cycling Xenopus Extracts
   
   
2-3 L of 1X MMR, make night before, store at 20°C.
200mL de-jelly solution, 2% cysteine, pH to 7.8 with 0.9mL 10N NaOH.
750mL XB:
10mM K-HEPES
50mM sucrose
1X XB
11uL 10N KOH per 100mL to keep pH 7.7
250mL CSF-XB:
250mL XB (from above)
5mM EGTA
1mM MgCl2
100mL CSF-XB + PIs:
100mL CSF-XB (from above)
10 ug/mL LPC
2M Sucrose
LPC
Cytochalasin
40X Energy Mix
Sperm
Extract Fix:
60% Glycerol
1X MMR
1ug/mL Hoescht
10% Formaldehyde
Sperm Dilution Buffer (5X)
10X Calcium: 4mM CaCl2 in 1X Sperm Dilution Buffer
   
   
If the extracts will be spun onto coverslips, then also need:
1. Prepare extracts as is done normally.  (Murray, 1991;Desai et al., 1999;Chen and Murray, 1997)  Do not centrifuge faster than 10K for 10 min at 16 deg C in HB-4 rotor (or equivalent in mini-ultra).  Higher speed spins will not cycle as well (or so I have read). 
 
Fixation Buffer:
BRB80
20% Glycerol
0.5% TX-100
4% Formaldehyde
 
Cushion:
BRB80
30% Glycerol
 
-20°C MeOH
   
   
 
2. Supplement the extract with cytochalasin D, 1 X energy mix, 50 mM Sucrose and LPC.  The use of cytochalasin seems to help extracts cycle.  Although, since it is more expensive, I would use it for cycling extracts only, and not for normal CSF.
 
Making a CSF Extract
   
   
Collect eggs.
3. Bring aliquots of extract to room temperature for 1-2 minutes. Add sperm at a concentration of  less than 1,000 sperm/ul (this can be varied, but I have read to high can overwhelm the extract) and gently flick the tube to thoroughly mix them the sperm in the extractGood mixing is essential, but the extract is sensitive, so be careful.   
 
Wash eggs in MMR 2-3X.
4. Add 1/10 volume of cycling solution which contains 4 mM CaCl2, 100 mM KCl, 1 mM MgCl2. Again gently flick the tube to mix thoroughly.
 
Remove as much MMR as possible, and pour on dejellying solution.
5. At about 30-45 minutes the extracts will have exited morphologically.  This can be checked by squashing 1 µl under 3 µl of squash fix (MMR + ~10% formaldehyde + ~50% Glycerol + Hoechst).  The chromatin will appear rounded and diffuse and they should be uniform throughout the extract.  That is, all nuclei should be at almost the same point.   
 
De-jelly 7-10 minRinse 1X in de-jelly. While eggs are de-jellying, add 1mL CSF-XB +PIs to centrifuge tubes.  Add 10uL 10mg/mL cytochalasin and flick tube well.
6. Check the extract every 10-15 minutes on the microscope.
 
Wash de-jellied eggs 3-4X in XB.   
7. When the chromatin is again condensed (around 90-120 minutes) add back an equal volume of room temperature CSF extract to re-arrest the cycling extract in mitosis. After -15-20 minutes check to be sure that the chromatin is condensed in a in a tight mass.
 
Wash eggs 2-3 X in CSF-XB.
8. Add nocodazole (if required for experiment) to a final concentration of 10 µg/ml (dilute nocodazole in extract).
 
 
Wash eggs 2X in CSF-XB + PIs.  Leave in small volume of CSF-XB+PIs.
9. Continue to incubate at room temperature for 15 minutes.
 
Gently pipet eggs into centrifuge tubes, put pipet below meniscus.
References
 
Remove excess buffer.
      1.    Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
 
      2.    Desai, A., A.Murray, T.J.Mitchison, and C.E.Walczak. 1999. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61:385-412.
Spin 10 sec at 1500 rpm (setting 4) in clinical centrifuge/
      3.    Murray, A.W. 1991. Cell cycle extracts. Methods Cell Biol. 36:581-605.
 
Aspirate off buffer. Add 100uL Versilube to top
 
Pack eggs: spin at 2000rpm (setting 5) for 30 sec and full speed for 1min in clinical centrifuge.
 
Aspirate off versilube and buffer from top of eggs.
 
Centrifuge packed eggs at 10,000 rpm for 15 min at 16°C in the SW55.1 rotor.
 
Collect cytoplasmic layer of extract with 18-guage needle into a 1-cc syringe. Avoid bubbles.
 
Transfer to new tube and estimate volume.
 
Add 1/1000 volume LPC.
 
Add 1/1000 volume cytochalasin.
 
1/40 volume 40X energy mix.
 
Add 1/40 volume 2M sucrose.

Revision as of 08:53, 15 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


Mike’s Cycling Xenopus Extracts


1. Prepare extracts as is done normally. (Murray, 1991;Desai et al., 1999;Chen and Murray, 1997) Do not centrifuge faster than 10K for 10 min at 16 deg C in HB-4 rotor (or equivalent in mini-ultra). Higher speed spins will not cycle as well (or so I have read).

2. Supplement the extract with cytochalasin D, 1 X energy mix, 50 mM Sucrose and LPC. The use of cytochalasin seems to help extracts cycle. Although, since it is more expensive, I would use it for cycling extracts only, and not for normal CSF.

3. Bring aliquots of extract to room temperature for 1-2 minutes. Add sperm at a concentration of less than 1,000 sperm/ul (this can be varied, but I have read to high can overwhelm the extract) and gently flick the tube to thoroughly mix them the sperm in the extract. Good mixing is essential, but the extract is sensitive, so be careful.

4. Add 1/10 volume of cycling solution which contains 4 mM CaCl2, 100 mM KCl, 1 mM MgCl2. Again gently flick the tube to mix thoroughly.

5. At about 30-45 minutes the extracts will have exited morphologically. This can be checked by squashing 1 µl under 3 µl of squash fix (MMR + ~10% formaldehyde + ~50% Glycerol + Hoechst). The chromatin will appear rounded and diffuse and they should be uniform throughout the extract. That is, all nuclei should be at almost the same point.

6. Check the extract every 10-15 minutes on the microscope.

7. When the chromatin is again condensed (around 90-120 minutes) add back an equal volume of room temperature CSF extract to re-arrest the cycling extract in mitosis. After -15-20 minutes check to be sure that the chromatin is condensed in a in a tight mass.

8. Add nocodazole (if required for experiment) to a final concentration of 10 µg/ml (dilute nocodazole in extract).

9. Continue to incubate at room temperature for 15 minutes.

References

     1.    Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
     2.    Desai, A., A.Murray, T.J.Mitchison, and C.E.Walczak. 1999. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61:385-412.
     3.    Murray, A.W. 1991. Cell cycle extracts. Methods Cell Biol. 36:581-605.