One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

Recombination cloning encompasses a set of technologies that transfer gene sequences between vectors through site-specific recombination. Due in part to the instability of linear DNA in bacteria, both the initial capture and subsequent transfer of gene sequences is often performed using purified recombination enzymes. However, we find linear DNAs flanked by loxP sites recombine efficiently in b...

PCR amplification of cDNA libraries for cloning and screening.

Screening a cDNA library is a common practice for cloning genes of interest in many laboratories. Success of screening is largely dependent on the abundance of cDNAs representing genes of interest and the availability of suitable probes. The procedure involves several rounds of screening to obtain true-positive clones, which might take a week or longer. Typically, each round of screening requir...

A novel one-step approach for the construction of yeast surface display Fab antibody libraries

BACKGROUND Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. RESULTS Within this study, we aimed at imple...

Rapid one-step recombinational cloning

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restrict...

One-Step Reverse Transcription PCR (RT-PCR). Fatemeh Mehrpooyan*