IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

help: nucleus prep

Tao Wei twei at mccoy
Sat Jan 1 17:01:30 EST 1994


Hi, everybody out there!  

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 used 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?  



More information about the Plantbio mailing list

Send comments to us at biosci-help [At] net.bio.net