Nuclei from Arabidopsis suspension culture

kloska at mpimg-berlin-dahlem.mpg.de kloska at mpimg-berlin-dahlem.mpg.de
Tue Dec 6 18:09:40 EST 1994


Dear colleageus 

I've to prepare nuclei from arabidopsis suspension culture.
The only protocol I've found is dealing  with whole plants, 
and goes as followes:

* Grind in polytron with buffer A (0.8 M sucrose;25 mM Tris pH 7.8;
  10 mM MgCl2), filter through cheesecloth  and  harvest pellet  at 
  5000 rpm in JA10.
  
* Resuspend pellet in buffer B (0.4 M  sucrose 25 mM Tris pH 7.8;
  40 mM  MgCl2)  and  centrifuge  through  75%  percoll  cushion 
  (buffer  B plus 75% percoll).
  
* Resuspendate and wash pellet in buffer C (0.4 M sucrose; 50 mM 
  Tris pH 7.8; 5mM MgCl2; 20% glycerol).
  
Is this protocol applicable for suspension culture ? Are there any
modifications recommendet, aspecialy  to  get  rid  of the  access  
pectine which  suspension  cells  produce ? 
Any  other  tips  of any kind from experienced plant nuclei prepa-
rators will be highly appreciated. 


Thanks in advance and best regards

-----------------+------------------------------------------------
sebastian kloska | (kloska at mpimg-berlin-dahlem.mpg.de)
                 | Institut fuer Genbiologische Forschung Berlin
                 | Ihnestrasse 63
                 | 14195 Berlin Germany
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