Journal: Journal of visualized experiments : JoVE

Article Title: Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

doi: 10.3791/59972

Figure Lengend Snippet:

Article Snippet: Autoclave it before use.Carbenicillin stock in water (100 mg/mL)AnyN/AFilter sterilize through a 0.22 μm filter before use.LB + carbenicillin agar plates (100 μg/mL)Agar: BD DifcoAgar: 214010Make LB agar plates as normal and add carbenicillin to 100 μg/mL before drying.E. coli strain carrying pJR487 (CEN-/ARS+ piggyBac-containing plasmid)N/AN/ARequest from Brem lab.Yeast peptone dextrose (YPD) liquid mediaBD DifcoPeptone: 211677, Yeast Extract: 212750Add filter-sterilized D-glucose to 2% after autoclaving.YPD agar platesAgar: BD DifcoAgar: 214010300 mg/mL Geneticin (G418)Gibco11811023YPD + G418 agar plates (300 μg/mL)Agar: BD DifcoAgar: 214010Make YPD agar plates as normal and add G418 to 300 μg/mL before drying.1M LiOAcAnyN/AFilter sterilize through a 0.22 μm filter before use.10X Tris-EDTA (TE) buffer (100 mM Tris-HCl and 10 mM EDTA)AnyN/AFilter sterilize through a 0.22 μm filter before use.Complete synthetic agar plates (24.1cm x 24.1cm) with 5-fluoroorotic acid (5-FOA) [0.2% drop-out amino acid mix without uracil or yeast nitrogen base (YNB), 0.005% uracil, 2% D-glucose, 0.67% YNB without amino acids, 0.075% 5-FOA]5-FOA: Zymo Research, Drop-out mix: US Biological, Uracil: Sigma, D-glucose: Sigm), YNB: Difco5-FOA: F9001–5, Drop-out mix: D9535, Uracil: U0750, D-glucose: G8270, YNB: DF0919Salmon sperm DNAInvitrogen1563201152% polyethylene glycol (PEG) 3350Sigma1546547Dissolve in water and filter sterilize through a 0.22 μm filter before use.Hybrid yeast strain JR507 (S. cerevisiae DBVPG1373 x S. paradoxus Z1, URA-/URA-)N/AN/ARequest from Brem lab.Water bath at 39°CAnyN/ADMSOAnyN/ALarge shaking incubators with variable temperature settingsAnyN/AYeast fungal gDNA prep kitZymo ResearchD6005Open in a separate window.

Techniques: Plasmid Preparation