Q1：克隆数少或不长克隆？

A1：

1) 引物设计有误。设计及合成引物时，注意反向引物的序列顺序;

2) 插入片段纯度差。对载体和插入片段进行胶回收纯化（目录号：GE706），电泳检测胶回收产物后再进行连接反应；不推荐多管样品过⼀个柱子的方式回收DNA，会造成更多的EDTA、胍盐等杂质残留，抑制反应；用于重组反应的胶回收产物请溶解于ddH₂O中;

3) 线性化载体或插入片段比例不佳。请严格按照说明书推荐的方法计算各组分用量，用琼脂糖凝胶电泳检测样品质量及浓度;

4) 感受态效率低。自制感受态随着保存时间的增加，效率可能会降低，建议尽量使用新鲜感受态或高效的商业化感受态。

Q2：多数克隆不含插入片段？

A2：

1) 克隆载体及片段中含有质粒背景。酶切制备线性化载体时，提高快速内切酶的使用量，延长反应时间，使用胶回收纯化酶切产物；制备插入片段时尽量使⽤预线性化质粒作为扩增模板，扩增产物进行DpnⅠ消化及凝胶回收纯化；

2) 相同抗性质粒污染。注意实验环境的清洁及背景质粒的消除。

Q3：克隆含有不正确的插入片段？

A3：PCR产物中混有非特异性扩增。推荐优化PCR体系，提高特异性，使用凝胶回收PCR产物。

Q4：菌落PCR无条带或大小不对？

A4：

1) 引物不正确。推荐使用载体的通用引物或至少使用一条通用引物进行菌P鉴定；

2) 假阴性结果（插入片段较大）。建议提取质粒为模板进行PCR验证或酶切验证。优化PCR体系、程序；

3) 重组失败。只有空质粒的条带，说明重组不成功，载体线性化不完全，建议优化载体及片段质量。

说明书：点击下载

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