Creating Lac- E. coli Mutants

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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?


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

Preparing Solutions

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.

Undiluted bacteria to put in UV Stratalinker

1. Prepare two small plates (found in drawer near the area with plastic “to be autoclaved” bins)

2. Label one plate: “800 uJ’

3. Pipette 750 uL of bacteria (x 2) into plate (1.5 ml total)

4. Cover with lid and put into tray to carry to Stratalinker

5. Expose the bacteria to 800 uJ of UV radiation

a. Make sure the “uJ” sign has a light next to it
b. Must remove the lid of each plate before placing it into the Stratalinker
c. Place plate at the center

Preparing MacConkey agar plates

1. Get 3 MacConkey agar plates for each of the following dilutions (so 9 plates in total)

a. 800 uJ, 100
b. 800 uJ, 10-1
c. 800 uJ, 10-2

2. Fill each plate with 4-6 beads to spread bacteria (beads are in large tubes overhead on lab table)

Preparing Tubes

1. Label two micro centrifuge tubes according to their radiation exposure amount and dilution amount:

a. 800 uJ, 10-1
b. 800 uJ, 10-2

2. Place tubes in micro centrifuge tray (found near the beads and pipette boxes)

3. Fill each tube with 900 uL using sterile PBS bottle with pump (0.9 mL setting)

a. Move tubes to mark your progress