General Protocol for Resistance Assays in Liquid Media

1. Transform plasmid DNA into BL21(DE3) competent cells and plate on agar plates with the appropriate antibiotic. Incubate the plates overnight at 37 ºC.
2. Inoculate a single colony from the agar plate to start growth cultures in 10 mL of sterile LB broth with antibiotic at 37 ºC.
3. Grow bacteria until the OD600 is within the range of 1.2 to 1.5 and dilute all cultures with fresh LB media containing antibiotics back to an OD600 of 1.0. Divide the culture into two 5 mL aliquots, where one is for the control experiment without ethidium bromide and the other is for the experiment with ethidium bromide.
4. Make a concentrated stock of ethidium bromide (5 mg/mL) in sterile water and add a final concentration 240 µg/mL of ethidium bromide into one of the two 5 mL fractions.
   Note that the addition of ethidium bromide defines time = 0.
5. For the control experiment, add the same volume of sterile water as the volume of ethidium bromide into the second 5 ml culture.
6. Measure the OD600 at time = 0 and record these values.
7. Measure the OD600 each hr until 6 hrs and record these values. An additional OD600 measurement can be taken at a longer time (e.g., 11.5 hrs). Make sure to dilute the cells by at least 3-fold to obtain an accurate OD600 measurement.
8. Plot time on the x-axis and OD600 on the y-axis.
9. Repeat the experiment in triplicate for estimating errors at each time point. One or more biological replicates should be acquired on a different day.

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