An Ro of 59 Angstroms is quite reasonable for a FRET experiment, as long as you expect the distance between donor and acceptor to be in that range, or closer.

I don't know how to label a plasmid with FITC, but assuming this is achievable, how do you plan to label it only in the region of the specific sequence of interest? If the label is nonspecifically incorporated throughout the plasmid, most of it will be too far away from a particular point in the sequence to act as a donor to a sequence-specific binding protein. As a result, the energy transfer efficiency will be very low.

Adam B Shapiro Hi Adam, Thank you for your answer. It seems that I have no way to FITC label a specific sequence of my plasmid using regular DNA probe labeling kits. I will label the entire plasmid with Label IT® Tracker™ Intracellular Nucleic Acid Localization kit using high doses to get the maximum base labeled as much as possible. This can label 1 base per 20-30 bases of the plasmid making the plasmid visible under a fluorescent microscope (at least the kit's figures show pretty bright signals). My plasmid is tiny (2.2 kb) and I think parts of my plasmid will be in the range of mCherry's energy transfer. The length of the DNA base is 3.4 A. So 34 bp will be in the range (17 bp from left and 17 bp from right) if I label 2 bases of 34 bp. Do you think 2 or 3 FITC molecules will be enough to excite mCherry????

In my experience, the fluorescence increase of the acceptor (when exciting the donor) is a less sensitive method of detecting energy transfer than the fluorescence decrease of the donor. However, in this case you have no choice but to use the acceptor fluorescence increase, since most of the donor fluorophores will be too far away from the acceptor to undergo significant energy transfer. The issue will then be whether you can detect the increased acceptor fluorescence against the much brighter (albeit blue-shifted) donor fluorescence.

Adam B Shapiro It makes sense. Thank you.