GeneExpressionLabProtocolandDatasheet_v2.docx - Gene...

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Gene Expression Lab Objectives 1. Describe the concept of gene expression. 2. Describe how prokaryotic gene regulation occurs at the level of transcription. 3. Explain the structure and function of an inducible operon. 4. Describe how an inducer (allolactose) and the lac repressor ensure the expression of the lac operon only at appropriate times. 5. Describe how glucose influences lac operon function. 6. Analyze the effect of inducers and repressors on lac gene expression. 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 dispose of them in the glass waste box. Lab Activity 1 : Determine the optimal growth conditions for LacZ expression. 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. Clearly label 13 microcentrifuge tubes to correspond with the reactions listed in Table 1. Pipet the appropriate E. coli culture, or unused media for the blank, into each microcentrifuge tube and then add the Lysis Reagent. Cap the tubes and mix by gentle flicking with your finger. Incubate the tubes at room temperature for 10 min. 2. While the E. coli is lysing, clearly label 13 glass tubes to correspond with the reactions in Table 1 Be sure that your label is up near the top of the glass tube so it will not interfere with absorbance readings. 3. Pipet the appropriate volume of Z Buffer into each of the 13 glass reaction tubes and the blank as calculated in Table 1 4. When the E. coli are done lysing, place the microcentrifuge tubes on ice. 5. Quantitatively transfer the contents of each of your lysis microcentrifuge tubes to the corresponding glass reaction tubes containing the Z buffer. What setting should you need on your micropipettor to do this? . . 1
6. Add the volume of ONPG stock solution you calculated in Table 1 the reaction in each glass tube, flick gently to mix, and incubate at room temperature to begin the reactions. Set a timer to count up and record the time when ONPG was added to each reaction tube in Table 2 .

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