Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast
نویسندگان
چکیده
منابع مشابه
Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast.
The cloning of DNA fragments is a fundamental aspect of molecular biology. Traditional DNA cloning techniques rely on the ligation of an insert and a linearized plasmid that have been digested with restriction enzymes and the subsequent introduction of the ligated DNA into Escherichia coli for propagation. However, this method is limited by the availability of restriction sites, which often bec...
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Over the past decade, the focus of cloning has shifted from constructing plasmids that express a single gene of interest to creating multigenic constructs that contain entire pathways or even whole genomes. Traditional cloning methods that rely on restriction digestion and ligation are limited by the number and size of fragments that can efficiently be combined. Here, we focus on the use of hom...
متن کاملGeneral method for plasmid construction using homologous recombination.
We describe a general method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with > 80% efficiency. Qua...
متن کاملRecombination-mediated PCR-directed plasmid construction in vivo in yeast.
We have extended the technique of PCR-directed recombination in Saccharomyces cerevisiae to develop a simple method for plasmid or gene construction in the absence of suitable restriction sites. The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid. Co-transformation into yeast results in homologous recombination at a position directed by the PCR oligonuc...
متن کاملRapid cloning by homologous recombination in vivo.
Cloning techniques are fundamental to molecular biology. Classically, recombinant plasmid and/or phage vectors are prepared in vitro, the transformation step only serving for amplification purposes. A different approach would exploit the natural intracellular enzymatic machinery to produce recombinant DNA molecules. Such techniques are well-known to mutagenize chromosomal DNA by e.g. transposon...
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ژورنال
عنوان ژورنال: Cold Spring Harbor Protocols
سال: 2015
ISSN: 1940-3402,1559-6095
DOI: 10.1101/pdb.prot085100