NUCLEI ISOLATION

Line:_______________

Date:_______________

1. Harvest, weigh tissue, to ice:________________ g.
2. Dump into N2 in ice bucket; break up with spatula/scissors (ca. 300 ml N2).
3. Dump into Waring blender; cover with cheesecloth, grind at maximum rpm for ca. 30 sec.; add N2 if needed.
4. In cold room; after N2 gone, add to ca. 200-300 ml MNIB w/ PVP, EtBr, while stirring with large stir bar.
5. Attach vacuum, 15" Hg, 15 min.
6. Polytron, 30 sec @ 8 setting.
7. Filter HD79, with vacuum.
8. Regrind residue with MNIB, PVP, EtBr.
9. Filter HD79.
10. Combine filtrates; filter HD40.
11. 10 min at 1000 g; SS34 or GSA bottles.
12. Resuspend in NSB; vortex if necessary.
13. 10 min at 1000 g.
14. Resuspend in NSB w/ Triton X-100; vortex if necessary; hand homogenize.
15. 10 min at 1000 g.
16. Repeat 2 more times, pellet should be non green; repeat once more if necessary.
17. Resuspend with 3 ml NSB; vortex if necessary.
18. Add 3 ml PDM.
19. Add Proteinase K to 200 g/ml (fresh).
20. Incubate 18 hrs. at 37 C.
21. 20 min at 12,000 rpm; save pellet in case.
22. To supernatant do 3 phenol:chloroform extractions.
23. Dialyze 48 hrs, 0.1X NTE; 1 liter; change at 16 hrs.