NUCLEI ISOLATION

Friday, November 07, 2003

Description
NUCLEI ISOLATION FROM PLANTS

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

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NUCLEI ISOLATION