Rapid cloning by homologous recombinationin vivo
نویسندگان
چکیده
منابع مشابه
Rapid 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...
متن کاملGenerating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination
A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from t...
متن کاملan improved homologous recombination method for rapid cloning of the antibody heavy chain gene and its comparison with the homologous recombination and traditional cloning methods
background: the homologous recombination (hr) is one of the conventional cloning methods for the production of recombinant dna. it is a quick method for in vivo dna cloning without using the high cost restriction enzymes. a few modifications in the cloning procedure can increase the efficiency of this method. m aterials and methods: in this study, effect of heating on the rate of the igg1 heavy...
متن کاملRestriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.
In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactio...
متن کاملIn vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining.
The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences ...
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ژورنال
عنوان ژورنال: Nucleic Acids Research
سال: 1993
ISSN: 0305-1048,1362-4962
DOI: 10.1093/nar/21.15.3601