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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 2632.6. Media and Plates1. Plates for growing bacteria (100 mm), Luria Bertani medium containing 50 µg/mLampicillin.2. Defined minimal yeast medium: all minimal yeast media, liquid, and plates usedin this protocol are based on the following ingredients, which are sterilized byautoclaving for 15–20 min: 6.7 g/L yeast nitrogen base-amino acids (Difco 0919-15),20 g/L glucose, or 20 g/L galactose plus 10 g/L raffinose, and 2 g/L appropriatenutrient “dropout” mix (see in next paragraph). For plates, 20 g Difco spark MDMIbacto agar (Difco 0140-01) are also added.A complete minimal nutrient mix includes the following: 2.5 g adenine, 1.2 gL-arginine, 6 g L-aspartic acid, 6 g L-glutamic acid, 1.2 g L-histidine, 1.2 g L-isoleucine,3.6 g L-leucine, 1.8 g L-lysine, 1.2 g L-methionine, 3 g L-phenylalanine, 22 g L-serine,12 g L-threonine, 2.4 g L-tryptophan, 1.8 g L-tyrosine, 9 g L-valine, and 1.2 g uracil.To make liquid media or plates for selection of yeast expressing plasmid selectionmarkers or auxotrophic reporters, one or more ingredients are omitted from thecomplete minimal nutrient mix. Thus, “dropout medium” lacking histidine(denoted His − in the following recipes) would select for the presence of plasmidswith the HIS3, HIS5, double marker, and so on. Note: (1) the quantities of nutrientsdescribed above are enough to prepare 40 L of medium, which in most casesis more than will be required and (2) premade dropout mixes are available fromsome commercial suppliers.3. Specific yeast liquid media and plates for this protocol (100 mm).a. Yeast extract, peptone and dextrese medium (YPD) (rich medium): 10 g/Lyeast extract, 20 g/L peptone, and 20 g/L glucose, autoclave about 18 min.To make plates: add 20 g Difco bacto agar per liter of (unautoclaved) mix forliquid media, and autoclave about 18 min. 1 L makes approx 40 plates.b. Defined minimal dropout plates, with glucose as a carbon source: Trp − ; Ura −His − ; Ura − His − Trp − ; Ura − His − Trp − Leu − ; and Ura − His − Trp − Lys − .c. Defined minimal dropout media, with glucose as a carbon source:Ura − His − ;and Trp − .d. Defined minimal dropout plates, with galactose and raffinose as a carbonsource: Ura − His − ; Ura − His − Trp − Leu − ; Ura − His − Trp − Lys − ; Ura − His − Leu − ;Ura − His − Lys − ; and Ura − His − Lys − Leu − (this last, optional).e. Plates for growing yeast library transformations (240 × 240 mm 2 ): use minimaldropout plates (Trp − ), with glucose as a carbon source. Pour approx 250 mLmedium on each plate.2.7. Primers1. For cI-fusion plasmids: forward primer, to confirm correct reading frame 5′-ATGATC CCA TGC AAT GAG AG-3′.2. For LexA-fusion plasmids: forward primer, to confirm correct reading frame 5′-CGT CAG CAG AGC TTC ACC ATT G-3′.3. For JG4-5 plasmid: forward primer, FP1, 5′-CTG AGT GGA GAT GCC TCC-3′.Reverse primer, FP2, 5′-CTG GCA AGG TAG ACA AGC CG-3′.