Smolke:Protocols/Yeast Media Recipes

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Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe For 1 L Batch: 1.7g Yeast Nitrogen Base (stored in cold room) 5g (NH4)2SO4 (Ammonium Sulfate, stored in chemical stock cabinet, EMD 168355) Dissolve in 800mL Np H2O for YNB-AA stock Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang Autoclave (store at RT for media stocks) To use as working media: After autoclaving add: 100mL 10X (20%) Dextrose (BD 215530) 100mL 10X Drop Out stock (10X DO stock recipe here: [[1]]) 1 L Total Volume Yeast Nitrogen Base Minus Amino Acids Non-Inducing, Non-Repressing (YNB-AA NINR) Recipe For 1 L Batch: 1.7g Yeast Nitrogen Base 5g NH3SO4 Dissolve in 700mL Np H2O for YNB-AA NINR stock Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang Autoclave (store at RT for media stocks) To use as working media: After autoclaving add: 100mL 10X (20%) Raffinose (BD 217410) 100mL 10X (10%) Sucrose (Sigma S3929) 100mL 10X Drop Out stock 1 L Total Volume Yeast Peptone (YP) Media Recipe For 1 L Batch: 10g Yeast Extract [Bacto™ REF#212750] (stored in chemical stock cabinet) 20g Peptone [Bacto™ REF#211677] (stored in chemical stock cabinet) 80 mg of Adenine hemisulfate (Sigma Chemical Co. Ltd., cat. no. A-3159) (stored in chemical stock cabinet) Dissolve in 900mL Np H2O for YP stock Autoclave (store at RT for media stocks) To use as working media: After autoclaving add: 100mL 10X (20%) Dextrose 1 L Total Volume YPD (Yeast Peptone Dextrose) To pour any of these media stocks as plates: Add 20g agar per 1L final media volume before autoclaving See here [[2]] For yeast KanMX (G418) selection: Cannot use ammonium sulfate as primary nitrogen source Add G418 to YP or to YNB with 1 g/L MSG (Sigma G1626) instead of ammounium sulfate Aqueous solutions stored frozen at -20 to -70 °C should be stable for approximately six months. Powder G418 sulfate (Geneticin) is stored in chemical room. Add 400mg/L for selection (in plates) Add 200mg/L for maintenance (in liquid culture media) For yeast counterselection: Add 1 g/l of 5-FOA to YNB-SC solid media to counterselect for URA3 While adding 20% dextrose and 10x SC AA mix, have 5-FOA dissolved in DMSO and add before stirring and pouring out agar (for a typical single sleeve 500ml media batch, this can be 500mg of 5-FOA in 5ml DMSO) Add 1.3 g/l of 5-fluorocytosine (5-FC) to YNB-SC solid media to counterselect for FCY1 dissolve 2.6 grams into 200 ml, filter sterilize, add 50ml per 500ml sleeve of plates if stored cold or frozen, will crash out of solution; heating helps dissolve detailed useful compound info here: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/f7129pis.pdf For alpha-AAD/LYS2,5 counterselection replace nitrogen source with 2g/L (0.2%) D,L-alpha-aminoadipate (Bachem, F-1185) dissolve 1 gram alpha-AAD in 20ml of water and pH with KOH to pH 6.0 (otherwise insoluble), filter sterilize and add to YNB with special AA mix two recipes have been shown to work for this counterselection YNB-alpha-AAD + URA,TRP,HIS,LEU,LYS YNB-alpha-AAD + URA,TRP,HIS,LEU,LYS,MET,ADE special media informed by Gottschling lab site: http://labs.fhcrc.org/gottschling/Yeast%20Protocols/lore.html -Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (aided by Andrew Babiskin)