Rapid and Efficient Cosmid Cloning

Nucleic Acids Res. 1981 Jul 10;9(13):2989-98. doi: 10.1093/nar/9.13.2989.

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.

MeSH terms

  • Animals
  • Cloning, Molecular*
  • DNA Restriction Enzymes
  • DNA, Recombinant / metabolism*
  • Drosophila
  • Genetic Vectors*
  • Molecular Weight
  • Plasmids

Substances

  • DNA, Recombinant
  • DNA Restriction Enzymes