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1、New Studier media for auto-induction and other applications Reference: F.W. Studier (2005) Protein production by auto-induction in high-density shaking cultures. Prot. Exp. Pur. 41, 207-234. ZYM-5052 medium for auto-induction It is recommended to use this medium for auto-induction. The amount of kan
2、amycin (when appropriate) used should be 100 g/ml. 958 mlZYtryptone1% yeast extract0.5% 20 ml50 x MNa2HPO425 mM KH2PO425 mM NH4Cl50 mM Na2SO45 mM 20 ml50 x 5052glycerol0.5% glucose0.05% ?-lactose0.2% 2 ml1 M MgSO4MgSO42 mM 0.2 ml1000 x trace elementstrace elements0.2 x ZYP-5052 medium for auto-induc
3、tion This medium should be used when stronger buffering is needed. The amount of kanamycin (when appropriate) used should be at least 200g/ml. Better even would be 400 g/ml. 928 mlZYtryptone1% yeast extract0.5% 50 ml20 x P (formerly NPS)Na2HPO450 mM KH2PO450 mM (NH4)2SO425 mM 20 ml50 x 5052glycerol0
4、.5% glucose0.05% ?-lactose0.2% 2 ml1 M MgSO4MgSO42 mM 0.2 ml1000 x trace elementstrace elements0.2 x ZYM-505 medium for plasmid preparation This medium is used to grow high-density cultures for plasmid preparations. 968 mlZYtryptone1% yeast extract0.5% 20 ml50 x MNa2HPO425 mM KH2PO425 mM NH4Cl50 mM
5、Na2SO45 mM 10 ml100 x 505glycerol0.5% glucose0.05% 2 ml1 M MgSO4MgSO42 mM 0.2 ml1000 x trace elementstrace elements0.2 x MDG non-inducing medium This medium is used for growing working cultures (such as overnight pre-cultures) and freezer cultures (glycerol stocks). 955 mlsterile water 20 ml50 x MNa
6、2HPO425 mM KH2PO425 mM NH4Cl50 mM Na2SO45 mM 12.5 ml40% glucoseglucose0.5% 10 ml25% aspartateaspartate0.25% 2 ml1 M MgSO4MgSO42 mM 0.2 ml1000 x trace elementstrace elements0.2 x Stock solutions ZY1% tryptone10 g/L 0.5% yeast extract5 g/L Dissolve tryptone and yeast extract in the necessary amount of
7、 water to make up 1 L of total medium (e.g. 958 ml for ZYM-5052). 50 x M1.25 M Na2HPO4-7H2O335 g/L 1.25 M KH2PO4170 g/L 2.5 M NH4Cl134 g/L 0.25 M Na2SO435.5 g/L Add in sequence to 700 ml water and stir until all salts have been dissolved. Occasionally crystals appear but they can be re-dissolved in
8、the microwave. The pH of a 20-fold dilution in water should be 6.75. 20 x P1.0 M Na2HPO4-7H2O268 g/L 1.0 M KH2PO4136 g/L 0.5 M (NH4)2SO466 g/L Add in sequence to 770 ml water and stir until all salts have been dissolved. The pH of a 20-fold dilution in water should be 6.75. 100 x 50550% glycerol500
9、g/L 5% glucose50 g/L Add in sequence to 570 ml water and stir until all components have been dissolved. 50 x 505225% glycerol250 g/L 2.5% glucose25 g/L 10% ?-lactose100 g/L Add in sequence to 730 ml water and stir until all components have been dissolved. Lactose is slow to dissolve. It may take two
10、 hours or more at room temperature. The process can be sped up by heating in microwave. 500 x MgSO41 M MgSO4-7H2O24.65 g/100 ml For 100 ml stock solution dissolve 24.65 g MgSO4 in 87 ml water. Stock solutions (cont.) 80 x G40% glucose40 g/100 ml To make 100 ml stock solution add 40 g glucose to 74 m
11、l water and stir until all glucose has been dissolved. It may take 45 min or more at room temperature. The process can be sped up by heating in microwave. 100 x D25% aspartate25 g/100 ml For 100 ml stock solution dissolve 25 g aspartic acid in 84 ml water and neutralize with 8 g NaOH (pH 7). 1000x t
12、race elements50 mM FeCl3 20 mM CaCl2 10 mM MnCl2 10 mM ZnSO4 2 mM CoCl2 2 mM CuCl2 2 mM NiCl2 2 mM Na2MoO4 2 mM Na2SeO3 2 mM H3BO3 Preparation of 100 ml 1000x trace elements stock solution (in 60 mM HCl). Prepare stock solutions of the salts (except FeCl3) as mentioned in the table below. Dissolve 0
13、.1 M FeCl3 in 50 ml of 100x diluted concentrated HCl. Add to 36 ml sterile water: 50 ml0.1 M FeCl3-6H2O2.70 g/100 ml 2 ml1.0 M CaCl2-2H2O15.8 g/100 ml 1 ml1.0 M MnCl2-4H2O19.8 g/100 ml 1 ml1.0 M ZnSO4-7H2O28.8 g/100 ml 1 ml0.2 M CoCl2-6H2O4.76 g/100 ml 2 ml0.1 M CuCl2-2H2O1.70 g/100 ml 1 ml0.2 M NiC
14、l2-6H2O4.76 g/100 ml 2 ml0.1 M Na2MoO4-2H2O2.42 g/100 ml 2 ml0.1 M Na2SeO31.73 g/100 ml 2 ml0.1 M H3BO30.62 g/100 ml Chemicals Compoundmol. weightcompanyorder number tryptoneSigmaT-9410 yeast extractSigmaY-1000 glycerol92.10Roth3783.1 glucose180.16RothX997.2 ?-lactose-H2O360.32Roth6868.2 Na2HPO4-7H2
15、O268.07SigmaS-9390 KH2PO4136.09SigmaP-5379 NH4Cl53.49Merck1145.1000 Na2SO4142.0SigmaS-6264 (NH4)2SO4132.1OLS2701316 MgSO4-7H2O246.48RothP072.2 aspartic acid133.1SigmaA-8949 FeCl3-6H2O270.30SigmaF-2877 CaCl2-2H2O158.2SigmaC-3881 MnCl2-4H2O197.91SigmaM-3634 ZnSO4-7H2O287.56SigmaZ-4750 CoCl2-6H2O237.95SigmaC-8661 CuCl2-2H2O170.48SigmaC-6641 NiCl2-6H2O237.72SigmaN-5756 Na2MoO4-2H2O241.9SigmaM-1003 Na2SeO3172.9SigmaS-5261 H3BO361.83Merck165.1000 Arie Geerlof6 February 2007
