Plant & Animal Genomes XIV Conference Plant & Animal Genomes XIV ConferenceJanuary 14-18, 2006
Town & Country Convention Center
San Diego, CA
Dilara Begum , Ron Meis , Merriann Carey
Construction of random-shear insert libraries is utilized for whole genome shotgun sequencing or sequencing of BAC, fosmid or cosmid clones. Normally, the DNA is sheared to generate smaller fragments which in turn are ligated to a vector. Achieving high-cloning efficiencies when constructing this type of library requires preparation of a highly dephosphorylated blunt-ended vector and complete conversion of the sheared ends of the inserts into blunt-ended, phosphorylated DNA. We will describe how two products from EPICENTRE can be used to speed-up and simplify both steps. APex Heat-Labile Alkaline Phosphatase is a novel alkaline phosphatase that dephosphorylates 5’-phosphates from protruding, blunt and recessed DNA ends using a 10 minute protocol. The enzyme is completely and irreversibly inactivated when heated at 70°C for 5 minutes. A 10 minute APex Phosphatase treatment of linearized vector resulted in greater than 99% reduction in colonies containing self-ligated vector. In addition, APex Phosphatase is active in a broad range of buffers and shows greater than 95% activity over a range of pH from 5.5 to 12. The End-It Kit, on the other hand, is used for the preparation of DNA that has been sheared, nebulized or restriction enzyme digested. An optimized blend of T4 DNA Polymerase and T4 Polynucleotide Kinase is used in a single-step conversion of DNA with 5’- or 3-’ overhangs to 5’-phosphorylated, blunt–ended DNA which ensures a high-cloning efficiency.