On 2 Oct 1996, Price, Carl A wrote:
>> In describing her experience in isolating nuclei from arabidopsis roots, Mary
> Williams cautions that
>> "the nuclear pellet lies over a starch pellet - be careful to resuspend the
> soft, easy -to -resuspend nuclei but leave behind the sticky, more opaque
> starch pellet."
>> It's been a while since we isolated nuclei from anything, but I recall that
> nuclei can be nicely separated from starch (and most everything else) by
> isopycnic sedimentation in gradients of Percoll (or other silica sols). The
> gradients should, of course, contain the same concentrations of solutes as in
> the isolation medium. The sedimentation coefficients of nuclei are very large,
> so that the time and speed of centrifugation should be just sufficient to
> sediment the starch.
>> --Carl Price
Although I have not isolated nuclei from roots I have purified
them from arabidopsis leaves and from tomato fruit. Percoll is indeed
helpful, however a large percentage of the nuclei will penetrate even 80%
percoll. To overcome this problem I have run the crude nuclei extracts
through a percoll step gradient; from top to bottom 40%:60%:80%:2.2M
sucrose. Most of the nuclei pellet at the sucrose:80% percoll interface,
however, some may be found pelleted at the bottom of the tube, as told by
DAPI staining. In both places I found starch crystals. I think the
original protocol is from Jim Lincoln when he was in Bob Fischer's lab.