Ala-Kokko L, Prockop D J
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
Matrix. 1990 Oct;10(5):279-84. doi: 10.1016/s0934-8832(11)80182-4.
A modified procedure for preparing cosmid libraries from genomic DNA is described. Genomic DNA was partially digested with a restriction endonuclease, and DNA fragments of appropriate size fractionated by agarose gel electrophoresis. A cosmid library was prepared, prescreened, and used to isolate gene inserts with previously published procedures. In one series of experiments, a modified cosmid vector containing stuffer fragments was used to prepare cosmid libraries containing partial SphI digests of 25 to 35 kb. From 10(5) to 10(7) clones were obtained per microgram of size-fractionated genomic DNA. From 10 to 100 hybridization-positive clones of a single copy gene (COL2A1) were obtained from plates that were positive in the prescreening step. Restriction mapping of over 20 clones and nucleotide sequencing of over 20,000 bp in each of two clones indicated that the inserts were faithful copies of the gene. In another experiment, a standard cosmid vector was used to prepare a cosmid library containing partial BamHI fragments of 30 to 45 kb. Genomic libraries can be prepared with 5 to 20 micrograms of genomic DNA and a large number of clones containing 25 to 45 kb fragments of a single copy gene can be isolated in about three weeks.
本文描述了一种从基因组DNA制备黏粒文库的改良方法。基因组DNA用限制性内切酶进行部分消化，合适大小的DNA片段通过琼脂糖凝胶电泳进行分级分离。按照先前发表的方法制备、预筛选黏粒文库并用于分离基因插入片段。在一系列实验中，使用一种含有填充片段的改良黏粒载体来制备包含25至35 kb SphI部分消化片段的黏粒文库。每微克大小分级的基因组DNA可获得10⁵至10⁷个克隆。从预筛选步骤呈阳性的平板中获得了10至100个单拷贝基因（COL2A1）的杂交阳性克隆。对20多个克隆进行限制性图谱分析以及对两个克隆中的每一个进行超过20,000 bp的核苷酸测序表明，插入片段是该基因的忠实拷贝。在另一项实验中，使用标准黏粒载体来制备包含30至45 kb BamHI部分片段的黏粒文库。用5至20微克基因组DNA可制备基因组文库，并且在大约三周内可分离出大量包含单拷贝基因25至45 kb片段的克隆。
