家蝇基因组文库的构建

Construction of Musca Domestica Genomic Library

从孵化 36 h的蝇蛆中提取基因组 DNA,用限制性内切酶 Bcl 随机酶切 ,回收 10～ 2 3kb的酶切 DNA片段 ,通过匹配粘性末端与磷酸化的 EMBL3Bam H 酶切载体臂连接 ,在体外经包装系统包装成活的重组噬菌体 ,成功地构建了家蝇基因组文库。重组噬菌体转染 KW2 51 宿主菌 ,测定文库效价为 5× 10 4 pfu/m

A Musca domestica genomic library was constructed, which was consisted of 1.2×10 5 recombinants with 15 kb average insert length ranging from 10 23 kb. High molecular weight genomic DNA with more than 50 kb size was extracted from the larva of fresh eggs hatched 36 h, which was digested with unfrequently cutting restriction enzyme BclⅠ. DNA fragments ranging from 10 23 kb were recovered by agarose gel electrophoresis loaded with digestion products of genomic DNA, which were ligated with EMBL 3 BamHⅠ Arms CIPase treated. Ligated DNA was packed in vitro using packing protein, later the packaged recombinant phage were transformed to KW 251 to titer genomic library, the cloning efficiency was 5×10 4 pfu/mL.