Q：一步法克隆试剂盒中同源重组酶作用机制？

A：同源重组酶兼具外切酶活性、DNA 聚合酶活性及连接酶活性。 a）外切酶活性：该酶能特异性地识别载体和插入片段末端的 15-20bp 的同源序列， 并沿着 5'→3'方向切割dsDNA，从而产生粘性末端，粘性末端的碱基在 50℃作用下进行互补配对，将具备同源序列的载体与插入片段“融合”。 b）DNA 聚合酶活性：外切酶作用后，并不能保证将片段和载体的 5’末端都降解成一样长的粘性末端，同源末端退火配对后，还存在单链的碱基缺口。故需要借助DNA 聚合酶活性进行修复。 c）连接酶活性：催化形成磷酸二酯键，将所有缺口缝合。

Q：与传统克隆相比，Hieff Clone 克隆有什么优势？

A:优势：a）无需考虑酶切位点：不受插入片段酶切位点限制，适用于任何载体；插入片段兼容粘性或平末端。 b）设计简单：在插入片段的 PCR 扩增引物 5’端引入 20 bp 左右与载体末端同源的序列即可。 c）快速：50℃，20 min 即可完成重组。 d）应用广泛：可用于单片段或多片段定向克隆，也可结合Canace 高保真酶，应用于定点突变。

Q：哪里体现省时间了？为什么不用考虑酶切位点？

A:a）少了对目的片段酶切和回收步骤，且连接反应无需过夜，只需 20 min，相当于省去一天时间。 b）不用考虑酶切位点是指载体可以选择任意酶切位点进行线性化，无需考虑目的片段上是否含有相同的酶切位点。

Q：引物设计原则？设计引物可否不引入酶切位点？退火温度如何选择（Tm 值如何算）？

A:a）设计原则：同源臂（15-25 bp，GC 含量 40-60%）+酶切位点(根据实验需求保留或删除)+基因特异性引物（常规插入片段扩增引物）。 b）可以不引入。 c）计算扩增引物退火温度时，只需计算基因特异性扩增序列的 Tm 值，引入的同源序列及酶切位点不应参与计算。

Q：最佳克隆位点选择？

A:应避免选择克隆位点上下游 50 bp 内有重复序列的区域。并且克隆位点上下游 20 bp 区域内 GC 含量尽量在 40%-60%范围内。

Q：假阳性—不含插入片段（空载）？

A:原因 1：载体线性化不完全 判断方法：将已制备的线性化的载体转化涂板，如果长克隆较多，则表示线性化不完全。解决方案：若线性化不完全则需优化酶切体系，例如提高限制性内切酶使用量、延长酶切反应时间、胶回收纯化酶切产物等，具体优化操作可参考限制性内切酶供应商的使用说明（通常推荐双酶切）。 原因 2：反应体系中混入了相同抗性的环状质粒 产生原因：①以反向 PCR 方式进行载体线性化，残留环状质粒模板导致。 ②目的基因PCR 模板为与载体相同抗性的环状质粒，残留环状质粒模板导致。

判断方法：将已制备的线性化载体和目的片段分别转化涂板，如果长克隆较多，则表示有相同抗性的环状质粒混入。

解决方案：PCR 扩增产物先用DpnI 消化模板后，再进行胶回收纯化处理或直接进行胶回收纯化处理，去除残留的环状模板质粒导致的假阳性。

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