PREMISE OF THE STUDY An efficient alternative strategy to conventional cloning was needed to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies. This method would facilitate studies and minimize technical problems typically encountered in cloning methodologies. METHODS We tested a variety of single-strand conformation polymorphism (SSCP) protocols...

We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The express...

the aim of this study was to clone the serine alkaline protease-encoding gene from bacillus subtilis 168. this protease, which can have many applications especially in detergent, may be industrially an important enzyme. for the amplification of the gene, pcr was performed with a pair of primers specifically designed for this purpose. electrophoresis of the pcr product showed the expected band o...

Green fluorescent protein (GFP) has become a convenient and versatile tool as a reporter protein in many aspects of science. Here, we show that the enhanced yellow fluorescent protein (EYFP) variant may be used advantageously as a reporter system for directional cloning of blunt-ended PCR products. We have constructed a pUC18-derived plasmid containing a reporter gene coding EYFP cloned into th...

designing and producing a proper fusion construction is the most important problem of producing large quantities of a properly folded functional protein. this construction should have all necessary components of a real gene. a good designed fusion gene construction could be cloned into a good and suitable host. clostridium perfringens is an important pathogen of humans and livestock and produce...

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5' end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3' overhangs designed to specifically complement each other. The combin...

Background: Interferon-gamma [IFN-γ) is the most important cytokine in immune system. This protein has been expressed bacterial cells. However, cloning not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ overcome problems favor of commercial purposes. Materials Methods: To amplify gene product for cloning, we primarily designed two specific primers target gene. Foll...

Epitope tagging simplifies detection, characterization and purification of proteins. Gene fusion to combine the coding region of a well-characterized epitope with the coding region for a protein of interest generally requires several subcloning steps. Alternatively, a PCR strategy can be used to generate such a chimeric gene. In addition to its simplicity, this approach allows one to limit the ...

We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA ...

background: the advent of recombinant dna technology has facilitated heterologous expression of proteins from various sources in different host systems including escherichia coli. if a plant virus coat protein is expressed in the bacterium it can be used as the antigen for antibody preparation. such a recombinant antigen preparation can be particularly useful where equipment such as ultracentri...