PCR Amplification and Plasmid Isolation

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.