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

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


   
   
Mike’s Cycling Xenopus Extracts
Sara's Cycling Xenopus Extracts
   
   
To prepare: 5 days before the experiment, prime female frogs with 50 IU of PMSG.
   
   
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). 
The night before the experiment, induce ovulation by injecting the primed frogs with 500 IU of HCG.  Place each frog in 2L of 1X MMR overnight at 18°C.
   
   
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.
In the morning, collect laid eggs.  Place the frogs back into 1X MMR and continue to 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 extract.  Good mixing is essential, but the extract is sensitive, so be careful.
Once you have sufficient amounts of eggs, wash the eggs in 1X MMR to remove debris and bad eggs.
   
   
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 de-jelly the eggs with 2% cysteine solution prepared in 1X XB salts. Swirl eggs occasionally and de-jelly until the eggs pack tightly.  Pour off de-jelly, and rinse briefly with fresh de-jelly solution. 
 
Wash the eggs into 1X MMR.  Activate the eggs by addition of 2μg/mL A23187, a calcium ionophore. (To 10 mL MMR, add 1μL ionophore).  Watch for activation (takes about 10 minutes).  Upon activation, wash out ionophore liberally with MMR. Place the eggs at 18°C for 50 minutes.
   
   
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.   
Wash the eggs 4X with XB, and 2X with XB+protease inhibitors (10μg/mL each leupeptin, pepstatin and chymostatin (LPC)).  Load the eggs gently into ultraclear centrifuge tubes containing 1 mL XB+PIs and 100μg/mL cytochalasin B or D.   
   
   
6. Check the extract every 10-15 minutes on the microscope.   
Pack the eggs by spinning at low speed in a clinical centrifuge for 30-60 seconds. Aspirate off the buffer and spin at moderate speed in the clinical centrifuge for 30-60 seconds.  Aspirate off residual buffer.
   
   
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.
Crush eggs by spinning in the mini ultracentrifuge swinging bucket rotor (RP55S-344) at 10,000 rpm for 10 minutes at 2°C.
   
   
8. Add nocodazole (if required for experiment) to a final concentration of 10 µg/ml (dilute nocodazole in extract).
Remove the cytoplasmic fraction with a 1mL syringe and a 18-guage needle.
 
9. Continue to incubate at room temperature for 15 minutes.
   
   
References
Add 1/1000 volume of LPC, 1/1000 volume of cytochalasin B or D, and 1/40 energy mix and 1/40 sucrose to the cytoplasmic fraction.  Invert and flick gently to mix.
   
   
      1.    Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
The extract is now ready for use.
      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.

Revision as of 09:14, 14 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


Sara's Cycling Xenopus Extracts


To prepare: 5 days before the experiment, prime female frogs with 50 IU of PMSG.

The night before the experiment, induce ovulation by injecting the primed frogs with 500 IU of HCG. Place each frog in 2L of 1X MMR overnight at 18°C.

In the morning, collect laid eggs. Place the frogs back into 1X MMR and continue to collect eggs.

Once you have sufficient amounts of eggs, wash the eggs in 1X MMR to remove debris and bad eggs.

Remove as much MMR as possible and de-jelly the eggs with 2% cysteine solution prepared in 1X XB salts. Swirl eggs occasionally and de-jelly until the eggs pack tightly. Pour off de-jelly, and rinse briefly with fresh de-jelly solution.

Wash the eggs into 1X MMR. Activate the eggs by addition of 2μg/mL A23187, a calcium ionophore. (To 10 mL MMR, add 1μL ionophore). Watch for activation (takes about 10 minutes). Upon activation, wash out ionophore liberally with MMR. Place the eggs at 18°C for 50 minutes.

Wash the eggs 4X with XB, and 2X with XB+protease inhibitors (10μg/mL each leupeptin, pepstatin and chymostatin (LPC)). Load the eggs gently into ultraclear centrifuge tubes containing 1 mL XB+PIs and 100μg/mL cytochalasin B or D.

Pack the eggs by spinning at low speed in a clinical centrifuge for 30-60 seconds. Aspirate off the buffer and spin at moderate speed in the clinical centrifuge for 30-60 seconds. Aspirate off residual buffer.

Crush eggs by spinning in the mini ultracentrifuge swinging bucket rotor (RP55S-344) at 10,000 rpm for 10 minutes at 2°C.

Remove the cytoplasmic fraction with a 1mL syringe and a 18-guage needle.

Add 1/1000 volume of LPC, 1/1000 volume of cytochalasin B or D, and 1/40 energy mix and 1/40 sucrose to the cytoplasmic fraction. Invert and flick gently to mix.

The extract is now ready for use.

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