The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that ...

Background: Tuberculosis (TB) remains as a major cause of death around the world. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid and evaluation of its expression in eukaryotic cells. ...

conclusions the results of the present study will increase our knowledge about molecular cloning and expression of the l. infantum kmp-11 gene, and this may be used as an effective target for controlling visceral leishmaniasis. results the kmp-11 gene was successfully subcloned in pcdna3 as a eukaryotic expression vector. recombinant kmp-11 protein was confirmed by the reverse transcriptase pol...

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10(6) transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of ...

Advances in molecular and synthetic biology call for efficient assembly of multi-modular DNA constructs. We hereby present a novel modular cloning method that obviates the need for restriction endonucleases and significantly improves the efficiency in the design and construction of complex DNA molecules by standardizing all DNA elements and cloning reactions. Our system, named HomeRun Vector As...

Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, w...

Repetitive DNA sequences play an important role in biology, especially at the level of eucaryotic gene transcription control. Their construction, assembly and cloning by conventional means or by PCR derived methods is usually very cumbersome. This method is based on the association of a small DNA tag with the sequence to be polymerised. The multimeric sequence is constructed in a stepwise manne...

We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain ...

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...