凝胶内长片段基因克隆的实验方法研究

The Study of In-gel Gene Cloning Method for Large DNA Fragment

目的量少、片段长的目的基因受纯化回收效率低的影响而难于克隆,本实验的目的是探索快速高效克隆量少,片段长DNA的实验方法。方法以lnc RNA AL110200 5’RACE大片段PCR产物为目的基因,应用高纯度、高分辨率的低熔点琼脂糖,分离得到含5.0 kb和2.0 kb的凝胶,直接用于连接和转化反应,通过克隆PCR和Sanger测序法鉴定克隆的正确性。结果经测序验证了5.0 kb和2.0 kb重组克隆的正确性,说明应用高纯度的低熔点琼脂糖凝胶可以快速克隆长达5.0kb基因片段。结论该方法是一种省时、高效、易行的长片段基因克隆方法。

Objective It is difficult to clone large DNA fragments with low amount affected by low rate of recovery.The purpose of the study is to explore an improved method for large fragment DNA cloning.Methods The large target fragments was from 5’RACE PCR fragments of Lnc RNA AL110200,and the low-melting agarose gel with high purity was applied to separate the fragments of 5.0 KB and 2.0KB.The in-gel ligation and the transformation was accomplished and the colony was identified by colony PCR and Sanger sequencing.Results The colonies containing 5.0 KB and 2.0 KB were identified by Sanger sequencing.It could be applied for large fragment cloning by the high purity low-melting agarose gel without purification.Conclusion It is a simple,time-saver,high efficiency method for large fragment gene clone.