efficiency was not affected by DNA source or PCR condition. Blue colonies contained a blunt-end-ligated plasmid without insert. Twenty randomly chosen white colonies were sequenced. Nineteen of them contained inserts ended with a primer and an A/T pair formed by complementary overhangs, and one contained an abnormally formed blank plasmid that was missing a part of the original sequence. The nu...

with advent and development of dna recombinant technology and advantages of p. pastoris expression system, fusion (f) protein of pprv expression, because of effective immunodominant role could be an appropriate candidate for production of recombinant vaccine against ppr disease. in this study, f gene of pprv nigeria 75/1 strain (1637 bp) was amplified using rt-pcr and purified. it was then clon...

Objective(s): Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in humans and animals. Micronemes (MICs) are effective candidates for DNA vaccine. Materials and Methods: In this study, we evaluated the immune response of BALB/c mice against MIC3 gene of Toxoplasma gondii and interleukin 12 (IL-12...

We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PC...

Inverse fusion PCR cloning (IFPC) is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fu...

Background: Cholesterol oxidase (CHO) has various clinical and industrial applications. Recently, microbial CHO have received a great attention for their wide usage in medicine. Here, taxonomic characterizations of isolated strain from soil, optimization of the conditions for CHO production and biochemical characterizations of produced CHO enzyme were described. Finally, CHO gene was cloned int...

This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragme...