DNA measurements

"W. VANCE BAIRD -656-4953", 803, DEPARTMENT OF HORTICULTURE, CLEMSON UNIVERSITY, CLEMSON SC 29634 BAIRDW at CLEMSON.CLEMSON.EDU
Thu Apr 25 15:19:00 EST 1991


We are interested in using flow-cytometry to determine
DNA content in plant nuclei. We have had a difficult time
releasing nuclei from the cells (comparatively small <20 u,
and our physical disruption methods are inadequate) but we can make
tons of single cells (yes we are now trying chemical/enzyme
methods).  My question is: can we believe the fluorescence
readings that we acquire using whole, single cells??  We use
propidium iodide and excite at 488 or 514nm.  My concern
is that (i) the wall affects the readings; (ii) the chloroplasts,
we use leaf tissue, "autofluorese" in the red range when
excited by 488 nm light (and 514?) thereby over-estimating
DNA content of the nucleus; (iii) the chloroplasts/chlorophyll
absorbs in the red range and therefore underestimate
nuclear DNA content.  Any ideas, facts, thoughts as to the
whimsy or validity of using intact cells in such an effort??
Vance Baird (BAIRDW at CLEMSON.CLEMSON.EDU)
Horticulture Department
Clemson University
Clemson, SC 29634-0375
803/656-4953;  FAX 803/656-4960



More information about the Arab-gen mailing list