【摘要】 采用低熔点琼脂糖包埋红曲菌细胞核制成胶块,然后利用蛋白酶K消化被包埋的细胞核,纯化胶块里的大片段DNA,随机剪切法回收40 kb左右的DNA,与Fosmid载体连接,经包装、转染构建红曲菌的基因组Fosmid文库,库容量为3×104,平均插入片段长度为36.6 kb.将其中7 700个单克隆保存于21块384孔微孔板中,-80℃储存.该文库符合黏粒文库的品质要求,为克隆聚酮化合物基因簇或其他重要基因及其功能的研究奠定了基础.更多还原

【Abstract】 Megabase-sized DNA fragments were prepared by embedding Monascus nuclei in low melting-point agarose,lysis in SDS,and proteinase-K digestion.DNA fragments were then randomly cut and polished with a blunt-end repair kit.Fragments from 30～45 kb were ligated into the pCC1FOS vector,packaged into phage particles with lambda packaging extracts,and transfected into EPI300 E.coli cells,resulting in a genomic library with 3×104 clones.Restriction enzyme analysis revealed that the average insert is 36.6 kb in length.The resultant 7700 colonies were stored in 384-well microtiter plates at-80℃.This library is a typical Fosmid library,and will be useful for cloning polyketide synthesis gene cluster.更多还原