Re: isolation of plant nuclei

From: Derek Schulze (flow@post.queensu.ca)
Date: Wed Oct 04 2000 - 10:28:59 EST


In my humble opinion you are using too much Triton X.  You should scale
back to 0.1-0.2%.

Check the following reference out for a good protocol:
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Estimation of Nuclear DNA Content of Plants by Flow Cytometry, K.
Arumuganathan and E.D. Earle, Plant Mol. Biology Reporter Vol9(3)1991,
pg229-233
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I have used this protocol on a variety of species with great success with
the addition of a polyamine buffer from the following reference:

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R.J.Bino, J.N.De Vries, H.L.Kraak and J.G. Van Pijlen, Flow cytometric
determination of nuclear replication stages in tomato seeds during priming
and germination.Annals of Botany 69, 231-236, 1992
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At 07:03 PM 9/30/2000 +0200, Jaroslav Dolezel wrote:
>> "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

- Derek Schulze

Flow Cytometry and Confocal Microscopy Core Facility
Cancer Research Labs
353 Botterell Hall
Queen's University
Kingston, Ontario
K7L 3N6
Canada

http://meds.queensu.ca/medicine/crl/



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