Efficient oligo coupling of amino-11-ddUTP and postlabeling with NHS-ester dyes. (A) Diagram of the probe labeling workflow. A set of single-stranded oligonucleotides (primers) are first coupled to amino-11-ddUTP at their 3′ end using the TdT enzyme. After ethanol precipitation, amino-11-ddUTP coupled oligos are labeled with an NHS-ester conjugated fluorescent dye. After ethanol precipitation and column purification, the oligos are used for hybridization to cells and microscopic imaging. (B) PAGE analysis of the degree of labeling (DOL) of ddUTP and dye for oligos of different GC content. (Top panel) SYBR Gold channel in gray. (Bottom panel) The composite of the SYBR-channel (green) and the Quasar570 channel (red). (C) Quantification of B. (D) PAGE analysis of first and second step labeling efficiency for oligos with four different 3′ ends. (Top panel) SYBR Gold channel is in gray. The bottom panel shows a composite of the SYBR channel (green) and the Quasar570 channel (red). (E) Quantification of D.
