Creating Lac- E. coli Mutants
To create Lac negative E. coli mutants for student/faculty use.
1. Do you know how to use the spectrophotometer? The stratalinker?
2. Do you have enough PBS? Do you have enough bacteria?
Plate strain S-401 (Lac+, uvr phr (-)) from the -80 freezer onto a MacConkey or LB agar plate. Incubate at 37 degrees.
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.
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.