黑龙江镜鲤基因组DNA文库的构建

Construction of genomic DNA library of the mirror carp

纯化镜鲤体组织的高分子量基因组DNA,经限制性内切酶Sau3A Ⅰ部分消化后,10％-40％蔗糖密度梯度离心分离并回收9—23kb的DNA片段。以λDASHⅡ为克隆载体,与9—23kb的DNA片段进行连接和包装,构建了镜鲤的基因组DNA文库。随机选取5个独立的噬菌斑,提取DNA后用EcoR Ⅰ与BamH Ⅰ进行酶切分析,证明克隆插入率为100％。经测定,该文库的滴度是108pfu/mL,根据公式N=ln(1-P)/In(1-f),99％的镜鲤基因组包含在该基因组文库中。以鲤IGF-Ⅱ基因探针对该基因组文库进行Southem杂交,筛选到相关的阳性克隆,证明这是一个较为完整的镜鲤基因组DNA文库。

High molecular weight genomic DNA from the mirror carp was extracted and partially digested with Sau3A Ⅰ.9- 23kb DNA fragments were collected by 10% -40% sucrose gradient centrifugation and ligated into λDASH Ⅱ vector. Recombination DNA was packaged. The construction of the genomic DNA library in mirror carp was completed. 5 separated plaques were selected randomly. The extracted DNA was analyzed by the digestion of EcoR Ⅰ and BamH Ⅰ . The results show the recombination rate is 100% . The titer of the library is 108pfu/mL and according to the formula N = ln(l - P)/ln(l - f) , the genomic DNA library consists 99% genome of mirror carp. The Southern blot were carried out with the probe of common carp IGF - Ⅱ in the library. Many positive clones proved that this is a completed genomic DNA library of mirror carp.