Protocol of a Seamless Recombination with Specific Selection Cassette in PCR-Based Site-Directed Mutagenesis
نویسندگان
چکیده
منابع مشابه
Construction of a recombinant vector for site-directed mutagenesis in Salmonella typhimurium
BACKGROUND: Among all common techniques in sitedirectedmutagenesis, λ Red recombinase system has beenwidely used to knock out chromosomal genes in bacteria. In thismethod, there is always the risk of DNA Linear digestion byhost's restriction enzymes that leads to the low frequency ofrecombination. OBJECTIVES:To overcome this, we constructeda recombinant vector to disrupt phoP gene in Salmonella...
متن کاملA simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis
BACKGROUND Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥ 1...
متن کاملSite-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination.
Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence eleme...
متن کاملApplication of a Seamless and Restriction Endonuclease-free Cloning Method to Produce Recombinant Full-length N-terminal His-tagged Streptolysin O in E.coli
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...
متن کاملApplied an Efficient Site-directed Mutagenesis Method into Escherichia coli
A new technique for conducting site-directed mutagenesis was developed. This method allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of ß-galactosidase. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. In this paper, in order to deve...
متن کامل