(A) λ DNA can be replicated in a microfluidic flow cell using a licensing mix, containing HSS, followed by a replication mix, containing HSS and NPE. Adding a second replication mix containing the CDK inhibitor p27^kip prevents excessive origin firing. Digoxigenin-dUTP labels nascent DNA, which can be immunostained [22]. (B) Real-time imaging of replication forks in extracts with Fen1-mKikGR, which binds nascent DNA [25], and adaptation of this system to visualise the fate of labelled nucleosomes reconstituted on λ DNA [26]. The right hand panel shows a kymogram, a stack of images from a single position of interest, with a growing replication bubble marked by Fen1-mKikGR. After the right hand replication fork reaches the labelled nucleosome four different histone fates are observed. (C) Summary of the KERHMIT assay developed in [33]. The bottom panel shows an example kymogram, where the right hand replication fork collides with a stable leading strand DPC. After initial pausing at the DPC, CMG is shown to bypass the DPC, with the fork rate slowing down until possible recoupling to polymerase. Abbreviation: DPC, DNA–protein cross-link.