Ish-Horowicz D, Burke J F
Nucleic Acids Res. 1981 Jul 10;9(13):2989-98. doi: 10.1093/nar/9.13.2989.
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.
我们介绍了一种黏粒克隆方法，该方法可实现32kb至45kb的单个DNA片段的快速高效克隆。通过对克隆载体pJb8进行适当处理，我们制备了不能自我连接但能接受去磷酸化插入DNA片段的左右两端载体末端。插入片段通过用MboI或Sau3A进行部分消化产生，并进行去磷酸化处理以防止非连续片段的连接和插入。该方法无需对插入DNA片段进行大小测定，并可防止形成含有短插入片段或多个插入片段的克隆。1微克目标果蝇DNA可产生约5×10⁵个克隆，平均插入片段大小为38kb。我们还描述了一种制备质粒和黏粒DNA的快速高效方法。
