Directional cloning of blunt-ended PCR products.

作者：M P Weiner · 发表于：PubMed · 年份：1993 · 被引用次数：15 · 研究领域：Molecular Biology Techniques and Applications、RNA and protein synthesis mechanisms、Viral Infectious Diseases and Gene Expression in Insects

A method that allows the directional cloning of blunt-ended polymerase chain reaction (PCR) fragments is described. One PCR primer must be 5' phosphorylated. Extra bases are not required on either PCR primer. A linearized vector is enzymatically processed to contain a single 5'-terminal phosphate. The monophosphorylated vector is amenable to recombinant-insertion during ligation when the fragment is in the correct orientation. Increased recombinant yield results from incubating the monophosphorylated vector with a restriction enzyme (SrfI) that relinearizes nonrecombinant plasmids during the ligation reaction.