Hi, I have first immobilized the biotinylated strand onto the streptavidin-coated beads. Then I hybridized the complementary strand with a cy5 tag after the initial immobilization of the biotinylated strand. When I image them in a confocal microscope, I don't get any cy5 signal on the bead. What should I do?
Adam B Shapiro
Try to determine if the biotinylated strand is binding to the beads by preparing the same oligo with Cy5 dye attached at the other end.
Make sure the optical filters on the microscope are the correct ones for imaging Cy5.
Cy5 fluorescence may be getting quenched by the DNA on the other strand. It might be a good idea to put a spacer between the oligo and the DNA to get the Cy5 away from the DNA.
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