This chapter focuses on the transformation of the nuclear respiratory deficient mutants of yeast. The chapter describes the methods for (1) transformation of pet mutants with a plasmid pool and the isolation of plasmid(s) carrying the wild-type genes complementing the mutations; (2) transfer of the selected plasmid into Escherichia coli to amplify and purify the wild-type DNA insert for restriction analyses and sequencing; and (3) transformation of the pet mutant with a plasmid preparation from E. coli to confirm the presence of the wild-type gene copy in the insert of the isolated plasmid. The vector CV13 plasmid (YEp 13) has three components: pBR322, EcoRI fragment, and a PstI. The PstI fragment of nuclear yeast DNA contains the LEU2 gene that confers a means of positive selection for leu2 cells acquiring plasmid during transformation. To complete the identification of the CV13 plasmid isolated in E. coli as the carrier of the wild-type gene complementing a yeast nuclear mutation, a small-scale E. coli DNA preparation is used to transform the original pet mutant.
ASJC Scopus subject areas
- Molecular Biology