Department of Agricultural and Biological Engineering, The Pennsylvania State University, 249 Agricultural Engineering Bldg,, University Park, PA 16802, USA.
J Biol Eng. 2011 Dec 16;5(1):17. doi: 10.1186/1754-1611-5-17.
A modified BioBrick™ assembly method was developed with higher fidelity than current protocols. The method utilizes a PCR reaction with a standard primer set to amplify the inserted part. Background colonies are reduced by a combination of dephosphorylation and digestion with DpnI restriction endonuclease to reduce vector and insert background respectively. The molar ratio of the insert to vector in the ligation was also optimized, with the accuracy of the transformed construct approaching 100%.
开发了一种改良的 BioBrick™ 组装方法，其保真度高于当前的方案。该方法利用带有标准引物的 PCR 反应来扩增插入部分。通过去磷酸化和 DpnI 限制性内切酶消化的组合，降低了背景菌落，分别减少了载体和插入背景。还优化了连接中插入片段与载体的摩尔比，转化构建体的准确性接近 100%。
