摘要
为解决分子生物学方法中因纯化的新鲜材料不足而限制实验进展的情况,采用改进的克隆方法将目的DNA片断保存在菌株中。首先应用改进的DNA提取方法从赤潮甲藻塔玛亚历山大藻(Alexandriumtamarense)中提取总核酸,以提取的DNA为模板,优化PCR扩增条件,获得转录单元内间隔区(ITS)片段。将获得的ITS片段经SalI和PstI双酶切后与同样经过双酶切后的质粒载体pBluscriptSK+连接,转化受体菌XL1-Blue,克隆该DNA片段。该克隆方法简单易行,克隆效率完全可以满足一般实验要求。该克隆技术的应用为随时获得目的DNA提供一条途径。
To overcom the shortage of fresh materials demanded by molecular biological experiments,an improved cloning method was applied.Target DNA fragments were preserved in a strain of E.coli.Total nucleic acid from Alexandrium tamarense was extracted using a modified DNA extracting method.Using this DNA as template,the internal transcribed spacer(ITS)and5.8S ribosomal RNA gene(5.8S rDNA)region was obtained by an improved PCR condition.This ITS and5.8S rDNA region was digested with two restriction endonuclease enzymes Sal I and Pst I and linked to plasmid vector pBluescript SK + which was also digested by the same two restriction endonuclease enzymes,transforming this recombinant plasmid to the host,XL 1-Blue and obtaining the cloning DNA fragment.This cloning method is simple,practical and efficient able to meet the need for regular cloning in lab.This study can provide target DNA on call.
出处
《海洋科学》
CAS
CSCD
北大核心
2004年第12期49-53,共5页
Marine Sciences
基金
国家自然科学基金资助项目(30170499
40476059)
中国科学院知识创新重要方向性项目(KZCX2-211)
国家973计划项目(2001CB409701)
