Rapid and efficient cosmid cloning
1981; Oxford University Press; Volume: 9; Issue: 13 Linguagem: Inglês
10.1093/nar/9.13.2989
ISSN1362-4962
AutoresDavid Ish‐Horowicz, J. F. Burke,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoWe 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.
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