Background and Aims: DNA cloning, sub-cloning and site directed mutagenesis are the most common strategies in nearly all projects of recombinant protein production. The classical method of restriction site cloning is unsatisfactory due to the need for supply of restriction enzymes and the inefficiency of the digestion reaction. Many new methods, including recombinatorial cloning and ligation in...

Cloning of polymerase chain reaction (PCR) products can be a valuable research technique, but in practice the technical problems associated with the methodology may limit its usefulness. A variety of methods have been developed that facilitate PCR product cloning. These include blunt-end ligation cloning (5), ligation-independent cloning (1,6), introduction of restriction sites into PCR primers...

The pCaSpeR (1) and pUAST vectors (2) are two of the most commonly used Drosophila transformation vectors. However, although they have great utility in their current form, their multiple cloning sequences (MCSs) have a limited number of unique restriction sites (Figure 1). This is particularly true for the pUAST vector, whose MCS has only five unique sites. Further, neither of the MCSs in pCaSp...

BACKGROUND Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustra...

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system inclu...

Background: Human epidermal growth factor receptor 2 (HER2) is over-expressed in breast, ovarian, gastric, and prostate cancers used as a tumor marker the diagnosis of cancer. Monoclonal antibodies have been diagnostic therapeutic tool against HER2. Because difficulties associated with stability complexity construct high cost antibody production, we aimed to investigate, cloning, expression HER...

Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway clon...

Many different strategies are used for cloning polymerase chain reaction (PCR) products. Some use restriction sites pre-integrated into primers or contained in a generated fragment. Others, such as the one used by Stratagene (La Jolla, CA, USA) in its pCR-Script Direct SK(+) Cloning Kit, are based on a blunt-end ligation. These strategies require the use of additional enzymes to polish the end...

Chicken anemia virus was detected by PCR in tissue samples collected from poultry flocks in Gujarat,India. The VP1, VP2 and VP3 gene sequences of CAV from Anand, Gujarat were obtained after cloning thePCR products in pDrive cloning vector. Nucleotide sequence alignment with other CAV sequencesdemonstrated overall identity of 95-98.8%, 98.8-99.8% and 98.8-100% for VP1, VP2 and VP3 regions,respec...

background and objective: the outer membrane protein w ( ompw ) of vibrio cholerae is involved in stimulating the im- mune response via induction of protective immunity. it also plays an important role in bacterial pathogenesis by increasing the adaptability of pathogenic strains. in this study we aimed to clone v. cholerae ompw gene in the strain x-33 of pichia pastoris. materials and methods:...