A rapid DNA cloning system is a research interest of many scientists. TA cloning is one of the methods used for the cloning of PCR-amplified DNA molecules. The TA cloning method is a convenient and labor-saving replacement to traditional, restriction enzyme-mediated cloning strategies. A T-vector called pBlueskript ΙΙ SK-1 with the lethal gene ccdB was designed to construct a positive selection...

ethidium bromide, and nucleic acids are visualized on a UV transilluminator. Typically, about 50–100 ng of supercoiled pUC119-derived plasmid DNA, without any limitation concerning the length of the insert, can be detected using this method (Figure 1). This is sufficient to discriminate between transformants containing inserts of various lengths and the control DNA. Although some chromosomal DN...

Recombinant DNA vectors are fundamental tools in molecular biology and genetic engineering. One of the problems frequently encountered in vector construction is the lack of compatible restriction sites between the vectors and the DNA inserts. The conventional approach to solve this problem is to make the DNA fragments blunt-ended (3,4,12) or to add linkers or adaptors (10,11,14) before ligation...

The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed...

The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are ...

A 3#{189}-yr-old boy with infantile genetic agranulocytosis (IGA) has had recurrent infections since the age of 3 mo. Characteristically he shows permanent, almost complete neutropenia with relative monocytosis in the peripheral blood, together with a bone marrow rich in proliferative myeloid cells, but lacking in mature ones. Results of in vitro cloning of the patient’s bone marrow cells in so...

Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formatio...

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in develop...

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene ...