Publications

Methods in molecular biology (Clifton, N.J.). 2017-01-01; 1620.359-366.

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

Yonemoto IT, Weyman PD

PMID: 27709588

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.

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