Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Biotechniques. 2011 Feb;50(2):124-7. doi: 10.2144/000113514.
Bacterial cloning was first introduced over a century ago and has since become one of the most useful procedures in biological research, perhaps paralleled in its ubiquity only by PCR and DNA sequencing. However, unlike PCR and sequencing, cloning has generally remained a manual, labor-intensive, low-throughput procedure. Here we address this issue by developing an automated, computer-aided bacterial cloning method using liquid medium that is based on the principles of (i) limiting dilution of bacteria, (ii) inference of colony forming units (CFUs) based on optical density (OD) readings, and (iii) verification of monoclonality using a mixture of differently colored fluorescently labeled bacteria for transformation. We demonstrate the high-throughput utility of this method by employing it as a cloning platform for a DNA synthesis process.
细菌克隆技术早在一个多世纪前就已问世，此后成为生物学研究中最有用的程序之一，其普及程度可能仅次于 PCR 和 DNA 测序。然而，与 PCR 和测序不同，克隆通常仍然是一种手动的、劳动密集型的、低通量的程序。在这里，我们通过开发一种基于以下原理的自动化、计算机辅助的液体培养基细菌克隆方法来解决这个问题：（i）细菌的有限稀释，（ii）基于光密度（OD）读数推断集落形成单位（CFU），以及（iii）使用混合不同颜色荧光标记细菌进行转化来验证单克隆性。我们通过将其用作 DNA 合成过程的克隆平台来证明该方法的高通量实用性。
