30 nM (final concentration) of reconstituted CMG-Mcm10 complex (Figure 2—figure supplement 4) was preincubated with the 50 bp duplex fork substrate (0.5 nM) for 10’ in the absence of ATP in buffer containing 20 mM Tris Acetate pH 7.6, 5 mM DTT, 0.1 mM EDTA, 10 mM MgSO4, 30 mM KCl, 40 μg/ml BSA. The reaction was started by addition of 1 mM ATP and 40’ later unlabeled 50duplex LAG oligo was added (to 50 nM) as a trap for unwound DNA. The total reaction volume was 78 μl and at the indicated times, 11 μl aliquots were removed, stopped with buffer containing 20 mM EDTA and 0.1% SDS (final concentrations), and flash frozen in liquid nitrogen. Frozen reaction products were thawed quickly in room temperature water and separated on 10% native PAGE minigels. Gels were washed in distilled water, mounted on Whatman 3 MM paper, wrapped in plastic and exposed to a storage phosphor screen that was scanned on a Typhoon 9400 laser imager (GE Healthcare). The scanned gel was analyzed using ImageQuant TL v2005 software to obtain the quantitations shown in the graph below the gel.