ENZYMES SHOULD BE THE LAST THING ADDED TO THE TUBES, RIGHT BEFORE YOU BEGIN INCUBATION!! Enzyme Concentration Table Sample Buffer (pH 7) ONPG Enzyme 1. 3 ml 0.5 ml 0.5 ml (1:1000) 2. 3 ml 0.5 ml 0.5 ml (1:100) 3. 3 ml 0.5 ml 0.5 ml (1:10) Wait for 2 min. Sodium Carbonate Absorbance 0.5 ml -0.005 0.5 ml 0.003 0.5 ml 0.231 Sodium Carbonate Absorbance 0.5 ml -0.012 0.5 ml -0.024 0.5 ml 0.023 Substrate Concentration Table Sample Buffer (pH 7) ONPG Enzyme 1:10 tube 1. 3 ml 0.5 ml (1:1000) 0.5 ml 2. 3 ml 0.5 ml (1:100) 0.5 ml 3. 3 ml 0.5 ml (1:10) 0.5 ml Wait for 2 min. Temperature Table Sample Buffer ONPG Enzyme Wait for 5 min. Ice (4o C) Sodium Carbonate 0.5 ml 1. 3 ml pH7 0.5 ml 0.5 ml (1:10) 2. 3 ml pH7 0.5 ml 3. 3 ml pH7 0.5 ml Absorbance 0.302 0.5 ml (1:10) 37 o C 0.5 ml 0.209 0.5 ml (1:10) 65o C 0.5 ml -0.004 pH Table Sample Buffer ONPG Enzyme 1. 3 ml (pH 3.0) 0.5 ml 0.5 ml (1:10) 0.5 ml (1:10) 0.5 ml (1:10) 2. 3 ml (pH 7.0) 0.5 ml 3. 3 ml (pH 11.0) 0.5 ml Wait for 5 min. 37C Sodium Carbonate Absorbance 0.5 ml 0.116 0.5 ml 1.263 0.5 ml -0.020 ENZYMES SHOULD BE THE LAST THING ADDED TO THE TUBES, RIGHT BEFORE YOU BEGIN INCUBATION!!
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