Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination
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چکیده
منابع مشابه
Site-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...
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Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying...
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A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for site-directed mutagenesis of large (> 10 kb) plasmids. Application of this protocol to the generation of mutant rabies virus glycoproteins expressed by the baculovirus/insect cell system illustrates the usefulness of this approach in facilitating structure-function relationships in t...
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By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate...
متن کاملMethod of site-directed mutagenesis using long primer-unique site elimination and exonuclease III.
troducing undesired mutations during the preparation of the vector, can negatively affect the frequency of gene targeting in ES cells (13). In summary, the method described here offers an attractive alternative for the preparation of replacement vectors for gene targeting. This protocol can be used to perform site-directed mutagenesis in long genomic sequences cloned into plasmids with the high...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 2009
ISSN: 0736-6205,1940-9818
DOI: 10.2144/000113150