Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.
The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important needs of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformants at a frequency of approximately 0.1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0.18%. Non-selective transformation using this transformation protocol was shown to increase the frequency of gene disruption three-fold. Co-transformation showed that 30-40% of the transformation-competent cells take up more than one DNA molecule which can be used to enrich for integration and deletion events 30- to 60-fold. Co-transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2-5% the frequency of the single event.
Gietz
R D
RD
Department of Biology, University of Rochester, New York 14627.
Schiestl
R H
RH
D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D008094:Lithium / Q000494:pharmacology*; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D014170:Transformation, Genetic / Q000187:drug effects*
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D008094:Lithium