View source for Isolation of mitotic chromosomes from XTC cells (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]


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
 
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