| Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids. | |
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MedLine Citation:
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PMID: 22467364 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid. It can be applied successfully to small plasmids (up to 10 kb). However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers, we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction (PCR) cycles and 0.5 µl of polymerase (instead of 18 PCR cycles and 1.0 µl of enzyme in the standard protocol) were sufficient for the reaction. |
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Authors:
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Bogdan Munteanu; Mario Braun; Kajohn Boonrod |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Journal of Zhejiang University. Science. B Volume: 13 ISSN: 1862-1783 ISO Abbreviation: J Zhejiang Univ Sci B Publication Date: 2012 Apr |
Date Detail:
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Created Date: 2012-04-02 Completed Date: 2012-08-07 Revised Date: 2013-05-20 |
Medline Journal Info:
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Nlm Unique ID: 101236535 Medline TA: J Zhejiang Univ Sci B Country: China |
Other Details:
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Languages: eng Pagination: 244-7 Citation Subset: IM |
Affiliation:
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RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence Molecular Sequence Data Mutagenesis, Site-Directed / methods* Plasmids / genetics* Polymerase Chain Reaction / methods* |
| Comments/Corrections | |
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