PCR mutagenesis and gap repair in yeast

Abstract

Random mutagenesis of a gene and subsequent screening for phenotypic properties is a basic genetic procedure for studying gene function. When working with yeast, cloning mutated alleles into a yeast vector can be done directly due to the efficiency of homologous recombination. Here we give sample protocols for introducing random mutations to a gene using PCR and transformation of the resulting product into yeast. After screening resultant colonies for the desired phenotype, the plasmid containing the selected mutation is easily rescued to bacteria for sequencing. A reliable protocol for rescuing yeast plasmids to bacteria is given.