Nucleic Acids Research, 1981, Vol. 9, No. 13 2989-2898
© 1981
MOLECULAR BIOLOGY |
Rapid and efficient cosmid cloning
Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole Lane London NW7 1AD, UK
Received May 18, 1981. 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 dephosphorylated 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. 1µg of target Drosophila DNA gives about 5x105 clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.
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