The appropriate culture and the corresponding sugar

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the appropriate culture and the corresponding sugar from Table 3.1. Cap the tube and mix well. 3. Pipet 20 uL of each culture/sugar mixture into each microcentrifuge tube containing the unused culture media and lysis Reagent. Cap the tubes and mix by gentle flicking with your finger. Allow the bacteria to lyse by incubating the microcentrifuge tubes at room temperature for 10 min. 4. While the E. coli is lysing, clearly label 4 glass tubes to correspond with the reactions in Table 3.2 . Be sure that your label is up near the top of the tube so it will not interfere with absorbance readings. 2
Name:_____________________ Section:____________________ 5. Pipet the appropriate volume of Z Buffer into each of the 4 glass reaction tubes as calculated in Table 3.2 . 6. When the E. coli are done +lysing, place the microcentrifuge tubes on ice. 7. Quantitatively transfer the contents of each of your lysis reactions from the microcentrifuge tubes to the corresponding glass reaction tubes containing the Z buffer. 8. Add the volume of ONPG stock solution you calculated in Table 3 to each reaction, 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 4 . 9. When the reaction turns yellow (about the color of a Post-It note), stop the reaction by adding 1.0 mL of 1M sodium carbonate and record the time you stopped the reaction in Table 4. Gently flick the tube to mix, and store each tube on ice until all reactions are complete. Complete all reactions before beginning spectrophotometry. 10. Measure and record absorbance values as in Activity 1 and record your data in Table 4. Be sure to gently wipe off the cuvette with a Kimwipe before inserting the cuvette into the instrument. Calculate β-gal activity. 3
Name:_____________________ Section:____________________ Lab Activity 1: Table 1 Culture Conditions Rxn # Lysis Reagent E. coli Culture Volume Unused media for differences in enzyme Z buffer to bring to 2 mL total Vol. ONPG (4 mg/mL) for 0.8 mg LB only (plain media) 1a 20 µL 20 µL 180 µL 1b 20 µL 40 µL 160 µL 1c 20 µL 200 µL 0 LB+lactose (lactose culture) 2a 20 µL 20 µL 180 µL 2b 20 µL 40 µL 160 µL 2c 20 µL 200 µL 0 LB+glucose (glucose culture) 3a 20 µL 20 µL 180 µL 3b 20 µL 40 µL 160 µL 3c 20 µL

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