A rapid preparation of extracts for DNA replication in vitro.

A rapid preparation of cytoplasmic extracts using a small number of cells was developed for SV40 DNA replication in vitro. Compared with methods published previously, this new method has two advantages: First, cells of several human cell lines can be processed at the same time. Second, the time of preparation has been reduced from 10 h to 2 h by adding sucrose and reducing the concentration of NaCl in the dialysis buffer. Activities of extracts prepared from small numbers of cells with this new method to support SV40 DNA replication in vitro are high, reproducible and comparable to that of extracts from large numbers of cells with the methods traditionally used. These advantages will make it possible to study the regulation of DNA replication in irradiated or drug-treated human cells more efficiently. Therefore, this simple method should be a useful complement to the large-scale preparation for the general study of regulation of DNA replication in human cells.