Enzyme Lab.docx - Name_Megan Easterling Section_578 Enzyme...

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Name:_Megan Easterling Section:___578 ________ Enzyme Lab Objectives 1. Measure enzyme activity. 2. Determine how changes in substrate and enzyme concentration affect enzyme activity. Safety 1. Closed-toe shoes must be worn 2. Long hair tied back 3. Goggles must be worn at all times while experiments are being conducted in the classroom. Even if you’re done. 4. Gloves should be worn while performing the experiments. 5. All pipet tips should be ejected into the tip waste bin on your bench. 6. All ONPG should be poured into the waste container on the side bench. Remember to put the lid back on when you are done. 7. Rinse your glass cuvettes in the sink and place them in the glass waste box at the front of the room. Lab Activity 1: What are the effects of changing substrate concentration on enzyme activity? Complete Table 1 and Table 3 on your own before coming to lab. Calculate how much ONPG and Z buffer to use for each reaction. 1. Dilute the E. coli culture 1:5 by pipetting 200 µL of E. coli culture into a microcentrifuge tube. Add 800 µL of water, close the cap and mix well. 2. Clearly label 6 microcentrifuge tubes to correspond with the reactions listed in Table 1. Pipet the diluted Enzyme ( E. coli culture), or plain media for the blank, into each microcentrifuge tube and then add the appropriate volume of Lysis Reagent. Cap the tubes and mix by gentle flicking with your finger. Incubate the tubes at room temperature for 10 min. 3. While your E. coli is lysing, clearly label 6 glass tubes to correspond with the reactions in Table 1 . Be sure that your label is up near the top of the tube so it will not interfere with absorbance readings.

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