Fig. S7.

A microfluidic injection system to rapidly exchange components in a single-channel TIRF flow cell. A schematic of the system of PEEK tubing connected by PEEK switching valves that was used to remove free RecQ from the flow channel is shown. This system is connected upstream of the flow cell and downstream of the syringe pump. In this system PEEK tubing connects two 3-port “T” valves (valve 1 and valve 3), a 3-port “90°” valve (valve 2), and a 50-µL loop. The syringe is initially filled with SM buffer with 75 nM Sytox Orange. Using the sample loading configuration (Top), DNA stained with Sytox Orange is injected into the flow cell with valve 3 in the indicated position, allowing DNA to attach to the surface of the flow cell. The system is then set to the in-line configuration (Bottom), and the syringe pump is turned on. The resulting flow of SM buffer into the flow cell extends the attached DNA and allows attachment of the other end. After locating doubly tethered DNA, the system is put back into the sample loading configuration. A reaction solution containing RecQ, ATP, Sytox Orange, and ^AF488SSB is injected into the 50-µL loop, whereas the syringe is filled with a solution of ATP, Sytox Orange, and ^AF488SSB that lacks RecQ. The valves are set to the in line configuration and the syringe pump is turned on to push the 50-µL reaction solution within the loop into the flow cell to initiate unwinding by RecQ. Another 50 µL of the solution lacking free RecQ flushes the flow cell, thereby removing free RecQ protein.