linearity in ploidy screening

Jaroslav Dolezel (dolezel@risc.upol.cz)
Tue, 4 Jul 1995 11:20:28 +0200 (DFT)

Peter,
You are right, your weird results are almost certainly due to non-linear
relationship. To bring your CRBC peak from channel 90 to channel 100 (a
shift of 10 channels), you have to increase the gain by a factor of about
1.111. All peak positions (in linear mode) will be changed by this factor
(i.e. a peak on channel 180 will be shifted to channel 200 and not to
channel 190 as you suggest).

For large-scale ploidy screening, we calibrate the instrument using
nuclei isolated from a plant with known ploidy. Its peak position is then
periodically checked (beware of sample deterioration). With external
standardization, we try to control all variables (e.g. to ensure the same
dye concentration in all samples, we chop leaf tissues in LB01 buffer
containing the dye, one should be carefull when handling toxic dyes). We
also use the same type and amount of leaf tissue in all samples.

In doubt, we isolate and analyse simultaneously the nuclei from a plant
with known ploidy and from a "weird" ploidy plant. Fluorescence ratio of
their G1 peaks give us the answer. (Internal standardization is always more
precise, but also more laborious)

Happy ploidizing,
Jaroslav

Jaroslav Dolezel
Institute of Experimental Botany
Dept. Plant Biotechnology
Sokolovska 6
CZ-77200 Olomouc
Czech Republic
Tel.: +42-68-5228521
Fax: +42-68-5228523
Email: dolezel@risc.upol.cz

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