DNA Replication in Nucleus-Free Xenopus Egg Extracts

Extracts derived from Xenopus laevis eggs represent a powerful cell-free system to study eukaryotic DNA replication. A variation of the system allows for DNA replication not only in a cell-free environment, but also in the absence of a nucleus. In this nucleus-free system, DNA templates are licensed with H igh-S peed S upernatant (HSS) and then replicated with a concentrated N ucleoP lasmic E xtract (NPE). This method has the advantage of allowing replication of small plasmids with desired modifications and manipulation of the nuclear environment. This chapter describes the protocols needed to prepare HSS and NPE and how these extracts are used to study DNA replication.