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2 protocols using custom synthesized oligonucleotide primers

1

PCR Amplification and Plasmid Isolation

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PCR amplification with Phusion Hot Start II high-fidelity polymerase (Thermo Scientific, Waltham, MA) was performed according to the manufacturer’s manual using high-performance liquid chromatography (HPLC)- or PAGE-purified, custom-synthesized oligonucleotide primers (Sigma-Aldrich). Diagnostic PCR was done with DreamTaq (Thermo Scientific) and desalted primers (Sigma-Aldrich). DNA fragments obtained by PCR were loaded on gels containing 1% or 2% (wt/vol) agarose (Thermo Scientific) and 1× Tris-acetate-EDTA buffer (Thermo Scientific), excised, and purified (Zymoclean, D2004; Zymo Research, Irvine, CA). Alternatively, fragments were purified using the GenElute PCR Cleanup kit (Sigma-Aldrich). Plasmids were isolated from E. coli with the Sigma GenElute Plasmid kit (Sigma-Aldrich) according to the supplier’s manual. Yeast plasmids were isolated according to the methods described in reference 50 (link). Yeast genomic DNA was isolated using a YeaStar genomic DNA kit (Zymo Research). E. coli DH5α (18258-012; Invitrogen) was transformed chemically (T3001; Zymo Research) or by electroporation. Chemical transformation was done according to the supplier’s instructions. Electroporation was done in a 2-mm cuvette (165-2086; Bio-Rad, Hercules, CA) by using a Gene PulserXcell electroporation system (Bio-Rad), following the manufacturer’s protocol.
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2

Yeast Nitrogen Base Preparation

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Yeast nitrogen base without amino acids was purchased from Formedium. The dropout supplement was prepared from amino acids (Peptide Institute) and nucleotides (FUJIFILM Wako Chemicals). Custom‐synthesized oligonucleotide primers were obtained from Sigma‐Aldrich Japan. All other chemicals were of reagent grade. Deionized water was obtained from a Barnstead™ Smart2Pure™ Water Purification System (Thermo Fisher Scientific).
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