By David W. Burden, Donald B. Whitney (auth.)
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Extra resources for BiotechnologyProteins to PCR : A Course in Strategies and Lab Techniques
5 MORE ... 1 M p-nitrophenyl-a-D-galactoside, in water Spectrophotometer Micropipettes Microfuge tubes Method 1. Add 1 ml of yeast culture broth to a microfuge tube and pellet the yeast by centrifuging for 1 min at maximum speed. Transfer the supernatant to a separate tube and save. 2. Prepare six test tubes labeled 5, 10, 15, 20, 25, and 30 minutes. 5 buffer, 25 JlI ofYPG yeast broth supernatent, and 25 JlI p-nitrophenyl-a-D-galactoside. The reaction is initiated (time =0 minutes) when the substrate is added to the reaction mixture.
Cover the cells with iodine solution and incubate for 60 sec. Wash gently with water and drain. e. Cover the cells with 95% ethyl alcohol and incubate for 15 sec. Pour off and gently rinse with water. f. Cover the cells with the counter stain Safranine. Incubate for 30 sec and again rinse with water. Dry the slide on a paper towel or with blotting paper (don't wipe off the cells). g. Examine the cells at 400x to lOOOx under a microscope. E. coli are Gram negative and appear as pink cells. Gram positive cells are purple.
In this way, you will determine that the enzyme reaction is linear over the time frame used in the assay procedure. , the enzyme consumed the p-nitrophenyl-a-D-galactoside in less than 30 min, the graph would increase and then plateau. Typically, the enzyme assay is also studied at various enzyme concentrations to insure that the reaction is also linear over the enzyme concentrations used in the assay. The data is evaluated by plotting the absorbance of the assay (a constant incubation time) versus the increasing enzyme concentration.
BiotechnologyProteins to PCR : A Course in Strategies and Lab Techniques by David W. Burden, Donald B. Whitney (auth.)