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Overlap extension PCR and site-directed mutagenesis

JW Jeremy D. Woodward
IT Inga Trompetter
BS B. Trevor Sewell
MP Markus Piotrowski
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Overlap extension PCR was performed using KAPA HiFi DNA polymerase (PEQLAB Biotechnology) to construct chimeric enzymes according to the method of Wurch et al.40. Site-directed mutagenesis based on the QuikChangeTM protocol was performed with partially overlapping primers41 also using KAPA HiFi DNA polymerase. Primers were designed by attempting to fulfill the following criteria: a minimum of 8 non-overlapping 3′ bases; G/C at both ends; Tm > 64 °C; mutation > 4 bases from 5′ end and 6–8 bases from the 3′ end. Successful plasmid construction and mutagenesis was confirmed by DNA sequencing.

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