From: Al Sabirsh (alan@biogen.wblab.lu.se)
Date: Wed Sep 25 1996 - 07:29:01 EST
Robin Barclay wrote: > > Dear colleagues > We want to tag free DNA to follow its possible delivery into cells (cell lines > or human leucocytes) using flow cytometry (FACSort). We have been tinkering > with sytox green from Molecular Probes, but would welcome any suggestions for > other potential labels. The label should be excitable/readable in the above > system, and stay associated with DNA through a 80C warm-up step. > Robin Barclay > Scottish National Blood Transfusion Service > Edinburg Adding my two cents to the other suggestions: Boehringer Mannheim (among others) make a fluorescein labelled dUTP. This can be incorporated into DNA using PCR by adding it to the PCR mix (they sell a kit for this). You didn't mention where the DNA came from or whether or not you had constructed it yourselves. But if PCR can be used then you can label the DNA this way and transfect it into cells because it behaves more or less like normal DNA. I don't know if this would be sensitive enough though. Depends on the tranfection effeciency (which is often low). Another idea is to transfect your cells with the DNA of interest, fix them, permeabilize them and use fluorescent in situ PCR (with dUTP for example) to identify cells containing the sequence of interest. This assumes that you have PCR primers for your sequence (and thus knowledge of the sequence) and that you won't need live cells afterwards. The advantage is extreme sensitivity. Have a look at Patterson et al Science 1993 260:976-9 for an informative example (the paper is about RNA detection but its the same basically without the reverse transcriptase step). AL
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