Efficient procedure for site-directed mutagenesis mediated by PCR insertion of a novel restriction site
نویسندگان
چکیده
منابع مشابه
Site-directed mutagenesis using PCR-mediated introduction of silent mutations.
The alteration of a specific sequence of DNA using site-directed mutagenesis (SDM) provides an effective tool to probe the structure and function of gene products. With the advent of polymerase chain reaction (PCR), several new techniques were developed that facilitate SDM. An oligonucleotide containing mismatches at the desired site of mutation is used to generate a population of mutant fragme...
متن کاملConstruction 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 کاملAn efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol
BACKGROUND Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been...
متن کاملSimple version of "megaprimer" PCR for site-directed mutagenesis.
use in restriction-enzyme analysis. This procedure is cost-effective because it saves the use of expensive miniprep kits for use with only positively identified recombinant clones and limits typically time-consuming miniprep steps. Our method can be accomplished in a few minutes in two microcentrifuge tubes with minimal enzyme, expense, no phenol/chloroform extractions and no ethanol precipitat...
متن کاملModification of a PCR-based site-directed mutagenesis method.
Site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein’s structure and/or function. We wanted to generate mutants of human ETS1 cDNA in the pET15b vector (Novagen, Madison, WI, USA) from which we had been producing wild-type protein for structural studies (11). Since we were subject to the constraints of this vect...
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ژورنال
عنوان ژورنال: Plant Signaling & Behavior
سال: 2010
ISSN: 1559-2324
DOI: 10.4161/psb.5.12.13994