Flow cytometry questions

Pierre Lejeune Pierre.Lejeune at ulg.ac.be
Fri Sep 18 11:56:00 EST 1998


Hello to all,

I'd like to analyze DNA content of nuclei in seedlings of Arabidopsis by
flow cytometry. The machine I have access to is setup for detection of
PI (propidium iodide). My first try was rather dirty and the peaks were
not well resolved. I would like to know if anyone has a protocol for PI
that works in Arabidopsis. In particular, what is the best extraction
buffer, conditions for RNAse treatment, PI concentrations, pH, etc...
Here is the protocol I used previously:
- 10 seedlings were chopped with a razor blade in 100µl of ice-cold
buffer (20mM MOPS, 45mM MgCl2, 30mM sodium citrate, 0.1% Triton, pH 7.0)

- the volume was adjusted to 400 µl and the extract was filtered on a
60µm Nylon-net filter.
- 100µg of RNAse were added and the extract was incubated at room T for
30 min
- finally, 10 µg of PI were added 1 hour before analysis

Any comments or advice would be welcome.

Thanks for reading.


Pierre Lejeune

Dept Biologie Végétale
Univ. Liège
Belgium




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