John D. Gabel
jdg95005 at uconnvm.uconn.edu
Thu Sep 28 13:39:16 EST 2000
Hello. I am going to try to get through this without leaving too many bases
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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