(A) To validate the use of free biotin as a trap for displaced streptavidin, we examined the migration of the biotinylated leading strand ssDNA (50duplex LEAD single biotin, see Table 1) in the absence of streptavidin (lane 1), when biotin is added before streptavidin (lane 2), and when streptavidin is added without biotin (lane 3). The radiolabeled substrate was present at 0.5 nM and streptavidin and free biotin were at 2 μg/ml and 750 nM, respectively (final concentrations). The substrate was pre-incubated at 30°C for 5’ with biotin (lane 2), streptavidin (lane 3) or neither (lane 1) and then placed on ice for 10’. Then streptavidin was added to the reaction in lane 2 and all three reactions were incubated at 30°C for 10’ before loading on the gel. (B) The biotin trap does not displace streptavidin from biotin. As in (A), the substrate was pre-incubated with streptavidin before addition of free biotin and then incubated at 30°C. Samples were withdrawn from the reaction at the indicated times, quenched with SDS/EDTA and flash frozen in liquid nitrogen. The graph below the gel shows that dissociation of streptavidin is negligible over a 20’ time course in the presence of the biotin trap.