Dear Connie, I think this is the best way to really clog-up your machine: free undigested DNA is one big yelly pudding! What people do at Los Alamos is sizing DNA fragments (after cutting with restriction enzymes) by running them through a flow cytometer: however for molecular FCM you need a ultrasensitive machine with lower flow rate and single photon counting detection. Last article: cytometry 41, 203, 2000. Magnificent work, but not to be carried out on a commercially available machine: should this become available, I would be the first customer! Best regards, Dirk Prof. Dirk Van Bockstaele, PhD Laboratory of Hematology Head Flow Cytometry Antwerp University Hospital Wilrijkstraat 10 B-2650 Edegem Belgium phone 32 3 821 3900, fax 32 3 825 1148 > ---------- > Van: Connie[SMTP:cl538@columbia.edu] > Verzonden: vrijdag 12 januari 2001 23:12 > Aan: Cytometry Mailing List > Onderwerp: Facsing isolated DNA > > > I have someone here at Columbia who wants to do a two steps staining for a > DNA adduct with a 1' monoclonal antibody and a 2' FITC conjugated > antibody. > The major problem we have is that they have to isolate the nucleus from > cells with a nuclear isolation buffer, then treat the isolated nucleus > with > 2N HCL to unfold the DNA in order to expose the adduct. At last, they do > the staining to look for presence of the adduct in those HCL treated > nucleus > (the nucleus most likely are lysed already with only DNA remaining). > Does anyone have any recommendation in doing FACS with this type of DNA > staining (FACSing only DNA, that is not confined within a nucleus/cells). > I > would expect the size of the DNA to be smaller than debris, so what should > I > do with the FSC/SSC. And, how should I interpret the data, since I don't > even know whether I am looking at debris or DNA. >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:18 EST