help: plant nucleus prep

[Leonard N. Bloksberg]

In Article <2g6v3nINNcha at dns1.NMSU.Edu> "twei at spock (Tao Wei)" says:
> Hello, everybody  
> 
> Recently I am preparing nuclei from tomato leaves to get nuclear extracts.   
> As a start I followed a protocol from a friend. Leaf tissue is homogenized  
> in chilled homogenization buffer (10 mM MES pH 6.8, 10 mM NaCl, 0.25 M  
> sucrose, 5 mM EDTA, 0.2 mM PMSF, 0.15 mM spermine, 0.5 mM spermidine, 0.2%  
> Triton X-100) (1:3 w/v), filterd through 2 layers and 6 layers of  
> Miracloth respectively and then centrifuged at 2000x g for 10 min.  
> Precipitate is resuspended in 10 ml of homogenization buffer and  
> centrifuged again.  Initially this washing step can effectively separate  
> nuclei from chloroplasts.  After I ran out of the homogenization buffer I  
> had to prepare more following the recipe exactly the same as before.   
> Unfortunately the newly-made buffer could not allow the separation of  
> nuclei from chloroplasts, both nuclei and chloroplasts are precipitated. I  
> tried different sucrose concentrations from 0.25, 0.5, 0.75, and 1.0 M,  
> different pH from 6.0 to 7.0 and different salt concentrations from 10 to  
> 50 mM.  Even though I found pH is a factor of the most importance, no  
> unique pH can work as well as the homogenization buffer (pH 6.8) used  
> initially. It is so strange to me.  I do not have any idea about what is  
> going on there.  
> I also tried to use Percoll step gradient (10%, 30% and 60%, or 30%, 60%  
> and 85%) to separate nuclei from chloroplasts.  In all these trials,  
> chloroplast seems to be very heterogenous in density and distributed among  
> all three gradients although most chloroplasts floated on top of percoll.  
> I could not see any nuclear band. Could anybody out there have any  
> suggestions or working protocols to help me out?  
> 
I have never had this problem with tomato nuclei.  They always come clean
for me.  Since the purpose of the Triton X-100 is to lyse chloroplasts, the
fact that you have any chloroplasts left to co-migrate with your nuclei
suggests a problem with the triton to me.  I use 0.5% Triton.  Also, check
that your Titon is fresh.  I have used ancient bottles with no problem, but
it can't hurt to check.  I also try for 1:5 to 1:10 ration in the first 
grinding.  I assume you are following green color to see chloroplasts.
Good luck, and please drop me a note to let me know what you are doing with
your nuclei.  I may want to compare notes on some other things.
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.	Leonard N. Bloksberg
.	bloksber at pilot.msu.edu
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