From: Neal Benson (neal@cell.path.med.ufl.edu)
Date: Thu Apr 29 1993 - 17:32:24 EST
> > Does anyone have any experience running bird or lizard blood for > DNA content and/or sex determinations? We have been trying to use > a protocol with 0.1% sodium citrate + 0.1% Triton X-100 as the buffer > and staining with P.I. We have been having problems with the stability > of the fluorescence signal as the samples are being run. Any suggestions > or hints would be helpful. > While I do not have any direct experience with this, a user of my laboratory used the BD CycleTest reagent kit on blood from desert tortoises (not lizards but at least a reptile) and produced reasonable histograms with CV's in the 2-2.5% range on a FACScan. If you like to "home-brew" your reagents, rather than buying a kit, a similar formulation to the contents of the CycleTest reagents are given in Vindelov and Christensen, Cytometry 11(7): 753-770, 1990. Another possiblity for stability problems could be related to the character- istics of the instrument you're using. PI emission can be reduced by mixture with the volume of sheath fluid that occupies the sample tubing when you first start running your sample. As the sample runs, the sheath fluid is gradually flushed out by the sample itself, and the PI concentration becomes more constant. This can take up to 5 minutes or more on instruments with long sample introduction tubes, like the FACS sorters. Possible solutions to this problem are to (1) wait until stability is achieved, or (2) boost the sample to flush out the line, or (3) change the sheath buffer. I believe that the Vindelov & Christensen article mentions this, as well as other hints at achieving stability in DNA analysis. Though the focus of the article was on human samples, perhaps some of the material it discusses might be useful. -- Neal Benson | Phone: (904) 392-0008 Department of Pathology | FAX : (904) 392-4693 Box 100275 | Email: nbenson@nervm.nerdc.ufl.edu University of Florida | Gainesville, FL 32610 USA |
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