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@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
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:38 EST