Rapid and efficient cosmid cloning

作者：David Ish‐Horowicz, J. F. Burke · 发表于：Nucleic Acids Research · 年份：1981 · DOI：10.1093/nar/9.13.2989 · 被引用次数：1823 · 研究领域：RNA Interference and Gene Delivery、DNA and Nucleic Acid Chemistry、Advanced biosensing and bioanalysis techniques

We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.