To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-independent cloning (LIC) is well suited to rob...

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null i...

background: influenza viruses are a significant cause of morbidity and mortality. the influenza virus pandemics, 1918, 1977, and especially the most recent one, a/h1n1/2009, made evident the need for generating recombinant influenza h1n1 antigens which are essential to develop both basic and applied research programs. among influenza virus proteins, haemagglutinin (ha) is a major surface antige...

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...

conclusions this study can serve as a fundamental experiment for the construction of a ns3/ns4a eukaryotic expression vector and its expression in mammalian cells. further research is underway to evaluate the fragment immunogenicity in lab animal models. results the results showed that the fragment was successfully amplified and cloned into a eukaryotic expression vector. sequencing and enzyme ...

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...