Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids |
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| Authors: | Bogdan Munteanu Mario Braun and Kajohn Boonrod |
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| Institution: | 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. |
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| Keywords: | Site-directed mutagenesis (SDM) Mutant Plasmid |
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