Cycling extracts, sara's protocol (Stukenberg lab) and Isolation of mitotic chromosomes from XTC cells (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]


Purification of Mitotic Chromosomes-
Mitchison and Desai Method
   
   
Sara's Cycling Xenopus Extracts
   
   
DAY BEFORE


To prepare: 5 days before the experiment, prime female frogs with 50 IU of PMSG.
1. Arrest XTC cells (10-15 plates) with a final concentration of 1 µg/ml of Nocodazole (1µl per 10 ml of culture media) overnight (~16hrs).
2. Make up 10x Swelling Buffer (1x = 5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA,  pH 7.2).  Dilute a total of 150 ml to 1x, keeping most at RT and about 25 ml on ice. 
3. Chill HB-4 in centrifuge to 4°C.  Put 2 x 15 ml corex tubes on ice.  Chill 7 ml dounce homogenizer and tight fitting pestle on ice.  Thaw Hoechst. Make up solutions for sucrose gradient steps (see Buffers at end).  Turn on microscope in Burke Lab. 
4. Collect mitotic cells by mitotic shake / blow off.
5. Pellet cells by using Jouan CR4.12 centrifuge in 50ml tubes at ~2,000 rpm for 3 minutes.
6. Resuspend in 10 ml of RT  Swelling buffer.  
   
   
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.
7. After the pellet is resuspended, add 40 ml more of RT swelling buffer.
8. Swell at RT for ~5 minutes. 
9. Prepare lysis buffer described below and make up sucrose step gradient during swelling.
10. Pellet (same as above) and resuspend vigorously in 7 ml of ice cold lysis buffer (5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA,  pH 7.2 plus 0.1% Digitonin and LPC).  Excess digitonin is spun out (or settles out before use). 
11. Transfer the cells to a chilled 7 ml dounce homogenizer and disrupt with 15-20 strokes using a tight pestle, resting for 2 seconds after each dounce.
12. Add Hoeschst to 2µg/ml and check by fluorescence to see that spindles have been disrupted. 
13. Transfer to a 14ml round bottom, polypro falcon tube (#352029) and spin in an HB-4 at ~900 rpm for 1 min at 4°C to pellet interphase nuclei and debris.
14. Layer the supernatant (~6 ml) onto 30/40/50/60% (2ml, 2ml, 2ml, 3ml) sucrose step gradient (made up with in swelling buffer) in a chilled 15 ml corex tube. 
15. Centrifuge at 5k for 15 minutes and 4°C in HB-4 swinging bucket rotor, brake off.
16. Collect chromosome in flocculent white mass using a pasteur pipette from the interphase between layers of sucrose. 
17. Check chromosomes again with Hoechst.  If there are a lot of interphase nuclei still present spin gently (~500 x g, 30 seconds) in a chilled benchtop centrifuge.
   
18. Freeze mitotic chromosome in lN2.  
   
   
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 [[calcium ionophore|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. 
Buffers
   
   
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.
Swelling Buffer (1x):
5 mM PIPES, pH 7.2
5 mM NaCl
5 mM MgCl2
1 mM EGTA
   
   
Crush eggs by spinning in the mini ultracentrifuge swinging bucket rotor (RP55S-344) at 10,000 rpm for 10 minutes at 2°C.
Lysis Buffer:
5 mM PIPES, pH 7.2
5 mM NaCl
5 mM MgCl2
1 mM EGTA
 
LPC .1% Digitonin
   
   
Remove the cytoplasmic fraction with a 1mL syringe and a 18-guage needle. 
Stock Hoechst- 10 mg/ml
   
   
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.
Stock Nocodazole- 10mg/ml
   
   
The extract is now ready for use.
Coffee- Medium Roast + Half and Half + Sugar

Revision as of 09:31, 14 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


Purification of Mitotic Chromosomes- Mitchison and Desai Method


DAY BEFORE

1. Arrest XTC cells (10-15 plates) with a final concentration of 1 µg/ml of Nocodazole (1µl per 10 ml of culture media) overnight (~16hrs).

2. Make up 10x Swelling Buffer (1x = 5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA, pH 7.2). Dilute a total of 150 ml to 1x, keeping most at RT and about 25 ml on ice.

3. Chill HB-4 in centrifuge to 4°C. Put 2 x 15 ml corex tubes on ice. Chill 7 ml dounce homogenizer and tight fitting pestle on ice. Thaw Hoechst. Make up solutions for sucrose gradient steps (see Buffers at end). Turn on microscope in Burke Lab.

4. Collect mitotic cells by mitotic shake / blow off.

5. Pellet cells by using Jouan CR4.12 centrifuge in 50ml tubes at ~2,000 rpm for 3 minutes.

6. Resuspend in 10 ml of RT Swelling buffer.

7. After the pellet is resuspended, add 40 ml more of RT swelling buffer.

8. Swell at RT for ~5 minutes.

9. Prepare lysis buffer described below and make up sucrose step gradient during swelling.

10. Pellet (same as above) and resuspend vigorously in 7 ml of ice cold lysis buffer (5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA, pH 7.2 plus 0.1% Digitonin and LPC). Excess digitonin is spun out (or settles out before use).

11. Transfer the cells to a chilled 7 ml dounce homogenizer and disrupt with 15-20 strokes using a tight pestle, resting for 2 seconds after each dounce.

12. Add Hoeschst to 2µg/ml and check by fluorescence to see that spindles have been disrupted.

13. Transfer to a 14ml round bottom, polypro falcon tube (#352029) and spin in an HB-4 at ~900 rpm for 1 min at 4°C to pellet interphase nuclei and debris.

14. Layer the supernatant (~6 ml) onto 30/40/50/60% (2ml, 2ml, 2ml, 3ml) sucrose step gradient (made up with in swelling buffer) in a chilled 15 ml corex tube.

15. Centrifuge at 5k for 15 minutes and 4°C in HB-4 swinging bucket rotor, brake off.

16. Collect chromosome in flocculent white mass using a pasteur pipette from the interphase between layers of sucrose.

17. Check chromosomes again with Hoechst. If there are a lot of interphase nuclei still present spin gently (~500 x g, 30 seconds) in a chilled benchtop centrifuge.

18. Freeze mitotic chromosome in lN2.



Buffers

Swelling Buffer (1x): 5 mM PIPES, pH 7.2 5 mM NaCl 5 mM MgCl2 1 mM EGTA

Lysis Buffer: 5 mM PIPES, pH 7.2 5 mM NaCl 5 mM MgCl2 1 mM EGTA

LPC .1% Digitonin

Stock Hoechst- 10 mg/ml

Stock Nocodazole- 10mg/ml

Coffee- Medium Roast + Half and Half + Sugar

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