Wang Y, Wang H
Department of Radiation Oncology, Kimmel Cancer Center of Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Radiat Res. 1999 Jan;151(1):59-62.
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.
我们开发了一种使用少量细胞快速制备细胞质提取物的方法，用于体外SV40 DNA复制。与先前发表的方法相比，这种新方法有两个优点：第一，可以同时处理几种人类细胞系的细胞。第二，通过在透析缓冲液中添加蔗糖并降低NaCl浓度，制备时间从10小时减少到了2小时。用这种新方法从少量细胞制备的提取物在体外支持SV40 DNA复制的活性很高，具有可重复性，并且与传统方法从大量细胞制备的提取物相当。这些优点将使更有效地研究受辐射或药物处理的人类细胞中DNA复制的调控成为可能。因此，这种简单的方法对于人类细胞DNA复制调控的一般研究而言，应该是大规模制备方法的有益补充。
