Lab Protocols

Hahn Lab - Methods - EMS Yeast Mutagenesis EMS Mutagenesis of Yeast Linda Hoskins, Hahn Lab Last modified10/16/98 1. Grow a 50 ml O/N YPD yeast culture at 30 degrees C. 2. Determine cell density and transfer an amount of culture corresponding to 1 x 108 cells to several 15 ml conical tubes. Spin down cells and wash with 5 ml sterile water. Spin down cells and wash with 5 ml 0.1M sodium phosphate (NaH2PO4) buffer, pH 7.0. Spin down cells and resuspend in 1.7 ml buffer. 3. Transfer cells to glass culture tubes. In hood, add 50 ul EMS (ethyl methanesulfonate) (also have one control tube without EMS). Incubate on roller at 30 degrees for varying amounts of time, between 20 min. and one hour. 4. At each time point, add 8 ml sterile 5% sodium thiosulfate (autoclaved). This will stop the mutagenesis by inactivating the EMS. Each cell suspension should contain 1 x 107 cells/ml. Save an aliquot of cells at this point to use in figuring out the cell survival. * All glassware, plas...

Hahn Lab - Methods - Yeast Sporulation Sporulation and Dissection of Yeast a/alpha Diploids Linda Hoskins Last Modified Tue, Nov 6, 2001 These conditions work well for the Hieter yeast strains 1. Isolate diploid colonies. Inoculate 2 ml YPD + Ade with a single, large diploid colony and grow O/N at 30 degrees C to stationary phase. 2. Put 200 ul of stationary phase cells into 5 ml sterile, distilled H2O. Spin down 2 min. in clinical centrifuge. Wash again with 5 ml H2O. 3. Resuspend in 2 ml 0.5% (50 mM) KOAc (pH 7.0) + 0.5X nutrients for auxotrophic markers (usually Ade, Ura, Trp, Leu, His, Lys, Met). 4. Incubate on tube roller at rm. temp. for 3-7 days. 5. Check for sporulation under microscope. Spin down tetrads in a small glass tube and wash 3 times with 5 ml sterile H2O. Resuspend in 2 ml H2O. 6. Spin down 180 ul cells. Resuspend in 90 ul Zymolyase, 0.5 mg/ml in 1M sorbitol. Incubate at 30 degrees for 5-10 minutes. 7. Slowly add 0.3 ml H2O on ice to stop the rea...

Hahn Lab - Methods - Yeast Plates Yeast Plates Hahn Lab Last modified Tue, Oct 6, 1998 Amino Acid Mix (40X) Use 0.6 g/liter Tyrosine 2 g Arginine 4 g Serine 2 g Valine 2 g Threonine 4 g Isoleucine 2 g Phenylalanine 2 g Aspartic Acid 2 g Proline 2 g Glucose Complete Plates (1 liter): 1.7 g Yeast Nitrogen base -amino acids and ammonium sulfate 5.0 g Ammonium Sulfate 1 liter H2O pH to 7.0 using NaOH Add: 0.6 g Amino Acid mix (mix well before adding) 20g Bacto-agar (Difco) Autoclave 25 min. Add: 10ml (10/mg/ml) of any other required amino acids 10ml 0.4% Adenine sulfate in 0.1M HCl if required 10 ml 0.2% Uracil if required 50 ml 40% glucose YPD Plates (1 liter): Yeast Extract 10 g Bacto peptone 20g Bacto-Agar 20g H2O 1 liter Autoclave 25 min Add: 50 ml 40% glucose 5 ml 0.4% Adenine sulfate in 0.1 M HCl if required Add 10 ml 0.2% Uracil (only if plates will be used for tetrad dissection) 5-FOA plates (0.5 liter): Agar: 15 g Bacto-agar 300...

Hahn Lab - Methods - Method for spotting yeast cells for cell growth assay Method for spotting yeast cells for cell growth assay Ted Young, 2009 Fill wells of 96 well microplate with 240 microliters 10% glycerol. Add 60 microliters culture. Make five fold serial dilutions by removing 60 microliters of first dilution and pipeting into 240 microliters and so on. Insert yeast multi-pin apparatus into 96 well plate. Gently pull out being careful not to touch sides of the plate. Quickly put on Petri plate. This will give uniform plating of cells.

Yeast Transformation (high efficiency) Linda Hoskins/Hahn Lab Hahn Lab - Methods - DMSO LiOAc Yeast Transformation Last modified Wed, Jul 5, 2000 This more involved method gives higher transformation efficiency than the semi rapid method. Use DMSO if transforming a library to yeast and/or transforming a mutant strain which transforms poorly using the LiOAc method. 1. Inoculate 5 mls YPD with a single yeast colony. Grow O/N at 30 degrees C. 2. Add 0.5 mls culture to 4.5 mls fresh media, check A660. Add suitable amount of cells to 60 mls fresh media to give A660 = 0.2 (2 x 106 cells/ml). Grow to A660 = 1.0 (2 x 107 cells/ml), takes approximately 5 hours. 3. Spin down 50 mls cells, wash in 10 mls sterile water, re centrifuge, resuspend in 1 ml sterile water. Transfer to 1.5 ml sterile microfuge tube, spin down, resuspend in 1 ml sterile TE / LiOAc (made fresh from 10X TE [0.1M Tris-HCl, 0.01M EDTA, pH 7.5] and 10X LiOAc [1M LiOAc pH 7.5, adjusted with diluted acetic acid]). Sp...

Yeast transformation using lithium acetate (rapid method) Steve Hahn , Genetic Methods Last Modified December 1997 This method works well when transforming with a plasmid and is rapid. (for highest efficiency transformation, see DMSO transformation method) Grow 100 ml cells overnight in YPD to an A600 of ~1.0-2.0. (A small blob of cells from a plate inoculated into 100 ml YPD and grown for 14 hr at 30o is usually fine). Harvest cells (100 ml) by centrifugation (5K rpm for 5 min in GSA). If cell density is greater than 1.0, use correspondingly less cells. Resuspend cells in 5 ml TE pH 7.5 and transfer to 15 ml screwcap tube. Spin 2 min in clinical centrifuge. Resuspend cell pellet in 5 ml TE + 0.1 M lithium acetate. Spin 2 min in clinical centrifuge. Resuspend cell pellet in 2 ml TE + 0.1 M lithium acetate. Incubate on tube roller at 30o for 1 hr (can be incubated for up to four hours with no ill effects). Boil high molecular weight salmon sperm DNA for 3-5 min. Rapidl...