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ZJUS-B
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)  2012, Vol. 13 Issue (4): 244-247    DOI: 10.1631/jzus.B1100180
Communications     
Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids
Bogdan Munteanu, Mario Braun, Kajohn Boonrod
RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany
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Abstract  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.

Key words： Site-directed mutagenesis (SDM)      Mutant      Plasmid     
Received: 06 June 2011      Published: 06 April 2012
CLC:  Q319  
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Bogdan Munteanu
Mario Braun
Kajohn Boonrod
Cite this article:

Bogdan Munteanu, Mario Braun, Kajohn Boonrod. Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2012, 13(4): 244-247.

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http://www.zjujournals.com/xueshu/zjus-b/10.1631/jzus.B1100180     OR     http://www.zjujournals.com/xueshu/zjus-b/Y2012/V13/I4/244

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