JCVI: Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.
 
 
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Citation

Yonemoto IT, Weyman PD

Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

Methods in Molecular Biology (Clifton, N.J.). 2016 Oct 06; 1498. : 359-366.

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Abstract

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.