|
imported>Xenbase |
Line 1: |
Line 1: |
| Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]
| | ==Reporting a bug in Xenbase== |
| | If you find a bug in Xenbase, we would really like to know! |
| | However, it is very important that we can find your bug so that we can fix it. |
| | To help us find your bug, please fill out [http://xenbase.org/xenwiki/images/6/6e/Bug_report.txt this] form, and e-mail it to us! |
|
| |
|
| | | ==Bug Handling Process== |
| 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
| |
|
| |
| Coffee- Medium Roast + Half and Half + Sugar
| |