Rapid and efficient cosmid cloning

TLDR

The authors present a rapid and efficient cosmid cloning protocol for 32–45 kb fragments and a method for preparing plasmid and cosmid DNA. The protocol uses the vector pJb8 with engineered ends that cannot self‑ligate but accept dephosphorylated inserts generated by partial MboI or Sau3A digestion, preventing non‑contiguous ligation. The method obviates insert sizing, eliminates short or multiple‑insert clones, and yields ~5 × 10^5 clones per µg of Drosophila DNA with an average insert size of 38 kb.

Abstract

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.