Fast and efficient PCR cloning with pcmv-script PCR cloning kit
Tanya Hosfield Kerstien Padgett Tim Sanchez Quinn
Stratagene Cloning Systems, Inc.
Stratagene has expanded the applications of the PCR-Script cloning strategy by introducing a new vector for high-efficiency cloning of PCR fragments and expression in mammalian cells. The pcmv-script PCR cloning kit uses a PCR cloning method that features a one-hour ligation and doesnt require adding extra bases to the primers. In this method, the PCR products are incubated with Srf I-digested pCMV-Script vector, Srf I and T4 DNA ligase. Ligating insert and vector in the presence of T4 DNA ligase and Srf I enzyme reduces the background of self-ligated vector. We have tested the pCMV-Script vector with two different reporter genes and have confirmed high-level expression of the gene products in mammalian cells.
Cloning PCR-generated fragments using PCR-Script cloning kits is a popular method,1 proven to be efficient, easy and superior to other available methods.2 In this cloning method, PCR products are incubated with the SrfI-digested pPCR-Script vector, Srf I restriction enzyme and T4 DNA ligase. The Srf I enzyme recognizes the rare sequence 5-GCCCGGGC-3. By including Srf I enzyme in the ligation reaction, the PCR-Script cloning method maintains high concentrations of digested vector DNA and allows the use of nonphosphorylated, unmodified PCR products. The ligation efficiency of blunt-ended DNA fragments is increased by the simultaneous, opposing reactivity of the Srf I enzyme and the T4 DNA ligase on nonrecombinant vector DNA.
In many instances, researchers prefer to characterize a eukaryotic gene in
mammalian cells in order to analyze cor