Magjet ngs cleanup kit

We generated pCEN constructs coding for protein fusions with EGFP using Gibson assembly (Gibson et al. 2009) . First, we amplified the EGFP tag (insert fragment) from the plasmid pFA6-GFP(S65T)-HIS3MX using oligonucleotides targeting this ORF without selection markers. In parallel, we amplified the pCEN backbone with oligonucleotides targeting the ORF without stop codon (Supplementary Table S9). Both PCR reactions were digested with DpnI (NEB, USA) for one hour at 37°C and then purified using the MagJET NGS Cleanup Kit (Thermo Fisher Scientific, USA). 50 ng of the insert and 50 ng of the backbone (molar ratio of ~1:10) were adjusted to 2.5 µL and mixed with 7.5 µL of Gibson assembly master mix prepared accordingly with manufacturer instructions (NEB, USA). Reactions were incubated for 1 hour at 50°C. We transformed BW23474 (F-, ∆(argF-lac)169, ∆uidA4::pir-116, recA1, rpoS396 (Am), endA9(del-ins)::FRT, rph-1, hsdR514, rob-1, creC510) competent cells with 5µL of each reaction and selected on 2YT plates supplemented with 50 mg/L Kanamycin. We confirmed 8 colonies per transformation by colony PCR and subsequent Sanger sequencing of the purified plasmids. These confirmed plasmids were transformed into BY4741 competent yeast cells and selected on SC -ura media. Six independent colonies from each transformation were isolated for further analysis.