Root nuclear extracts

[Eric Brenner]

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. 
Eric Brenner