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NUCLEI ISOLATION

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.