Creating Lac- E. coli Mutants

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Goal

To create Lac negative E. coli mutants for student/faculty use.

Pre-Protocol Questions

1. Do you know how to use the spectrophotometer? The stratalinker?

2. Do you have enough PBS? Do you have enough bacteria?

Protocol

Day 1

Plate strain S-401 (Lac+, uvr phr (-)) from the -80 freezer onto a MacConkey or LB agar plate. Incubate at 37 degrees.

Day 2

Sterilely put 10 ml of LB into a sterile flask. Select one colony from the incubated plate, and inoculate the LB. Incubate in the shaking incubator at 37 degrees.

Day 3

1. Prepare solution of bacteria and sterile PBS (depending on date, the O.D might differ) in test tube with green cap

a. 1.5 mL bacteria + 1.5 mL sterile PBS

2. Use a blank containing at least 3 mL of PBS, insert into spectrophotometer, and press the arrow up button

3. Take out blank and insert test tube into the spectrophotometer

4. Check if OD is ~0.4

5. If OD is not ~0.4, remove 1 mL of the solution and add 1 mL of either PBS or bacteria depending on whether OD is higher or lower than 0.4. Repeat until OD is ~0.4.