碱性土壤基因组DNA的分离纯化和基因文库的构建

Extraction and purification of DNA from alkalescence environmental samples and construction of mixed genomic DNA libraries

直接从广西南宁市凤凰纸业排污沟碱性土壤样品中抽提和分离纯化混合基因组 DNA,所获得DNA的产量为每克土壤样品 1 0～ 3 0μg。采用限制性内切酶 Eco R1酶处理后 ,构建了以 p GEM-3 Zf(+)为载体的 DNA部分文库。文库的容量为 2 3 65 0个转化子 ,外源片段 DNA平均大小为 3 .2 kb。建库效率为每克环境样品获得 60 0 0个左右的含 1～ 1 5 kb外源随机插入片段的克隆。通过 DNA序列测定和同源性比较 ,对从文库随机调取的 1 6个转化子序列进行分析 ,发现 1 3个外源插入片段包含序列尚未确定的 DNA片段。

The mixed genomic DNA were extracted and purified from alkalescence environmental samples directly in this study. The DNA yields that have been got ranged 10 to 30 μg per gram of dry environmental sample. In this method, the mixed genomic DNA library was constructed by inserting restriction fragments of the purified DNAs into plasmids pGEM-3Zf(+) and then transferring into DH5α after the genomic DNA were digested with the restrictions enzyme EcoR1 partly. The library contained 23 650 positive clones and the average foreign fragment DNA was about 3.2 kb. And the efficiency of mixed genomic DNA was the approximately 6 000 inserting-containing clones which contained the fragment arranged from 1 kb to the 30 kb were obtained from 1 g dry samples. Clone libraries were analyzed by DNA sequencing and comparing with gene's identity. Among 16 randomly-selected clones,13 clones contained inserting fragments which sequences had not been confirmed.