Site-Directed Mutagenesis of Large Plasmids
نویسندگان
چکیده
منابع مشابه
Site-directed mutagenesis of large plasmids.
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...
متن کاملHomemade site directed mutagenesis of whole plasmids.
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...
متن کاملSite-directed mutagenesis of large (13-kb) plasmids in a single-PCR procedure.
Although there are many methods for site-specific mutagenesis, most are only applicable for short (< 7-kb) plasmids. The few methods that have been developed for longer plasmids are complicated procedures that involve multiple PCR steps or complex recombination and ligation reactions (2,4–6). Here, we provide a simple single-PCR procedure for site-specific mutagenesis of long plasmids (13 kb), ...
متن کاملEfficient method for site-directed mutagenesis in large plasmids without subcloning
Commonly used methods for site-directed DNA mutagenesis require copying the entire target plasmid. These methods allow relatively easy modification of DNA sequences in small plasmids but become less efficient and faithful for large plasmids, necessitating full sequence verification. Introduction of mutations in larger plasmids requires subcloning, a slow and labor-intensive process, especially ...
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...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 1998
ISSN: 0736-6205,1940-9818
DOI: 10.2144/98256st04