GSM6696603: △YRM11, Sort-seq; Saccharomyces cerevisiae; OTHER
Sort-seq: After measuring expression of library sequences with Sort-seq, plasmid DNA is extracted from yeast cells using EZ Yeast Plasmid Prep kit. Desired regions of the Sort-see library are amplified using PCR. Sort-Seq library was constructed using similar methods described in Lui et al., 2020. To prepare the vector plasmid pRM1806 for cloning, the plasmid was liberalized with high fidelity restriction enzymes KpnI and ApaI. We then purified the digest and   used a molar ratio of 3:1 plasmid to library DNA in a Gibson assembly reaction, following manufactures instructions. The Gibson assembly product was filtered by drop dialysis with a Nitrocellulose membrane (0.025 µm), following the Millipore Sigma protocol. The library product was electroporated into 7 0.1 cm (about 0.04 in) cuvette tubes, each containing 25 µl of E. coli electrocompetent cells and cells were then plated onto 28 15-cm Kanamycin containing plates. After 16 hours, plates containing 50,000 colonies each were scraped, and the plasmid DNA extracted using a Qiagen Maxiprep kit. 