Costa G L, Grafsky A, Weiner M P
Stratagene Cloning Systems, La Jolla, California 92037.
PCR Methods Appl. 1994 Jun;3(6):338-45. doi: 10.1101/gr.3.6.338.
Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.
本文介绍了提高PCR扩增DNA片段平端克隆产量及分析的方法。我们发现，用Pfu DNA聚合酶处理Taq DNA聚合酶扩增的片段可提高克隆的产量和效率。使用由两条外侧、距离不对称的引物和一条片段特异性引物组成的三引物组，可确定克隆插入片段的存在及其方向。应用这些方法，可在一天内完成片段的扩增和克隆，并在第二天对假定的克隆进行分析，从而节省大量的时间和精力。
