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CLC number: Q319

On-line Access: 2012-04-06

Received: 2011-06-06

Revision Accepted: 2011-09-29

Crosschecked: 2011-12-01

Cited: 5

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Journal of Zhejiang University SCIENCE B 2012 Vol.13 No.4 P.244-247


Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids

Author(s):  Bogdan Munteanu, Mario Braun, Kajohn Boonrod

Affiliation(s):  RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany

Corresponding email(s):   kajohn.boonrod@agroscience.rlp.de

Key Words:  Site-directed mutagenesis (SDM), Mutant, Plasmid

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

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%T Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids
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%A Mario Braun
%A Kajohn Boonrod
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T1 - Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids
A1 - Bogdan Munteanu
A1 - Mario Braun
A1 - Kajohn Boonrod
J0 - Journal of Zhejiang University Science B
VL - 13
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SP - 244
EP - 247
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1100180

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.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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