Protocol Transformation of Schizosaccharomyces japonicus Keita Aoki and Hironori Niki 1 Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka 411-8540 Japan This protocol describes the use of electroporation to transform Schizosaccharomyces japonicus with plasmids or linear DNA. Plasmids are helpful for the complementation testing of mutations and for the expression of specific genes. Linear DNA fragments integrated into chromosomal DNA by homolo- gous recombination are useful for gene deletion or to fuse a gene with a tag sequence (e.g., encoding a fluorescent protein). To introduce DNA into S. japonicus, electroporation methods are recommended because S. japonicus is sensitive to lithium acetate (LiOAc). MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Agar plates for selection of transformants (see Steps 14–15) DTT (1 M) Milli-Q water, sterilized and ice-cold Plasmid or linear DNA fragment (in distilled water or 1/2× TE buffer; see Step 8) Plasmids specific for transformation of S. japonicus (Aoki et al. 2010) were constructed using an autonomously replicating sequence (ARS) isolated from the S. japonicus genome. Schizosaccharomyces japonicus strain of interest Sorbitol (1 M) YE medium <R> YE liquid medium is required. If necessary, the medium can be supplemented with 0.1 mg/mL adenine and 0.1 mg/mL uracil (see Step 13). Equipment Electroporation cuvettes (0.2-cm), prechilled to 4 ˚ C We use those recommended for the Gene Pulser Xcell system. 1 Correspondence: hniki@nig.ac.jp From the Fission Yeast collection, edited by Iain M. Hagan, Antony M. Carr, Agnes Grallert, and Paul Nurse. © 2017 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot091850 996 Cold Spring Harbor Laboratory Press on May 29, 2020 - Published by http://cshprotocols.cshlp.org/ Downloaded from