View source for Cycling extracts, mike's protocol (Stukenberg lab)

Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]


link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]


 
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.