Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interferenc...

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LI...

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...

The efficiency of PCR product cloning depends on the nature of the DNA polymerase employed because amplicons may have blunt-ends or 3' adenosines overhangs. Therefore, for amplicon cloning, available commercial vectors are either blunt-ended or have a single 3' overhanging thymidine. The aim of this work was to offer in a single vector the ability to clone both types of PCR products. For that p...

An improved procedure for preparing PCR cloning vectors was developed. This procedure includes the incorporation of adapters to create XcmI restriction enzyme sites in pBluescript II SK(+) vectors, digestion with XcmI followed by further digestion of the small fragment produced by XcmI digestion with additional enzymes, and purification with PCR purification kits. Using this procedure, PCR clon...

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenes...

INTRODUCTION This protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to re...

BACKGROUND Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. RESULTS Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR a...

multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of rna interference (rnai) cassettes. although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. here, we worked out a simple cloning stra...