isolation of plant nuclei

Jaroslav Dolezel dolezel at aix.upol.cz
Sat Sep 30 12:03:58 EST 2000


Dear John,

unfortunately there is not a general procedure that will work with all
plants. When starting with a new material, we always test contrasting types
of isolation buffers to see which works best. In my lab, we use the
following buffers/procedures:

1) LB01 buffer (very good in preserving isolated nuclei; may result in
higher background and broader peaks in some species)
2) Tris-MgCl2 buffer (simple composition; surprisingly effective in some
species while completely failing in other)
3) Two-step procedure, or its simplified versions (Otto I and Otto II
buffers; may be more laborious but usually very effective)

Try to manipulate with the tissue/buffer ratio. The tissue should be CHOPPED
using a sharp razor blade and not just squeezed.

You will find the recipes, protocols and some useful comments on our web
pages http://www.ueb.cas.cz/olomouc1 and also in:
Galbraith, D. W., Lambert G. M., Macas, J., Dolezel, J.: Analysis of nuclear
DNA content and ploidy in higher plants. - In: Robinson, J.P.,
Darzynkiewicz, Z., Dean, P.N., Dressler, L.G., Orfao, A., Rabinovitch, P.S.,
Stewart, C.C., Tanke, H.J., Wheeless, L.L. (eds.): Current Protocols in
Cytometry. Pp 7.6.1 - 7.6.22. John Wiley & Sons, Inc., New York, 1998.

Good luck. Jaroslav

***************************************************************************
Dr. Jaroslav Dolezel
Laboratory of Molecular Cytogenetics and Cytometry
Institute of Experimental Botany
Sokolovska 6
CZ-77200 Olomouc
Czech Republic

Tel.: +420 68 5228521
Fax: +420 68 5228523

Email: dolezel at aix.upol.cz
Web site: http://www.ueb.cas.cz/olomouc1
***************************************************************************


> Hello. I am going to try to get through this without leaving too many
bases
> uncovered.
>
> I can not seem to isolate plant nuclei. I have tried several buffers and
> permutations of buffers (With/without spermine, w/w-o DTT, w/w-o RNAse,
> MOPS, HEPES, PBS etc.) I have run ranges of salt and pH around 6.8 to 7.5.
> I have tried boosting salt concentrations and lowering salt concentrations
> and so on. My plant of interest is an aquatic mustard very closely related
> to horseradish. It grows in rosettes and produces very small leaves (so
> obtaining the freshest youngest leaves are and issue) These leaves can get
> to be about 0.5 g of lamina when older.
> My protocol of greatest effect was to use a tris buffer of Pfosser et al
> (1995?). I chopped 0.75 g of leaf tissue in 3 ml of this buffer on ice and
> kept everything on ice. I let his incubate for 30 min. I got roughly 4-5
> nuclei/50microliters. These nuclei were very mangled looking and there
were
> many that were lysed (as viewed in phase under 400x).  When i then stained
> this prep with PI there were tons of starch grains staining and almost no
> nuclei (I could see chromosomes from lysed nuclei) I found only 2 intact
> nuclei in one prep of this........Now I know that most of you would say,
> "Raise the salt John!" But that has not seemed to help the situation. So,
> this buffer has .5% Triton X in it. Is that maybe too high?  What is a
good
> typical buffer that people use? (General guidline) What things do you play
> with other than salt concentration to optimize a buffer? There is no
course
> for me to take on this. I have read every book i can get my hands on. The
> guy who owns the FACscaliber i am using only looks at surface antigens in
> t-cells so he claims he can not help me.
>
> I realize that there are many things that I can be doing wrong. I am
unsure
> about what questions I should be asking. Can someone suggest and approach?
> Is there a big book of plant flowcytometric methods? Thanks in advance.
>
>
>
>John D. Gabel






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