A simple two-step method for efficient blunt-end ligation of DNA fragments.
A simple two-step method for efficient blunt-end ligation of DNA fragments.
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一种简单的两步方法，可有效实现 DNA 片段的平末端连接。
作者：
S. Damak;D. W. Bullock
重组质粒的形成是由线性化载体的一端与插入片段的一端之间的连接（高DNA浓度有利），随后是新产生的杂合分子的自连接（低DNA浓度有利）引起的。标准方案建议两种事件都可能发生的平均DNA浓度。由于该DNA浓度对于两个连接事件都不是最佳的，因此有效的平端连接受到损害。我们描述了一种平端连接的方法，在高DNA浓度下开始1小时，然后在1/20的初始DNA浓度过夜。用该方法获得的重组质粒的数量比用标准方案高约10倍。对两步连接法获得的10个重组质粒进行限制性酶切和琼脂糖凝胶电泳分析，结果表明所有质粒均含有一个拷贝的插入片段。
The formation of recombinant plasmids results from ligation between one end of the linearized vector and one end of the insert (favored by high DNA concentration), followed by self-ligation of the newly created hybrid molecule (favored by low DNA concentration). Standard protocols recommend an average DNA concentration at which both events may occur. Since this DNA concentration is not optimum for both ligation events, efficient blunt-end ligation is compromised. We describe a method for blunt-end ligation starting at a high DNA concentration for 1 h then at 1/20 the initial DNA concentration overnight. The number of recombinant plasmids obtained with this method is about 10-fold higher than with standard protocols. Restriction digestion and agarose gel electrophoresis of 10 recombinant plasmids obtained with the two-step ligation method showed that all plasmids contained one copy of the insert.