Inoculating Liquid Bacterial Culture

Collect all necessary materials including isopropanol, LB media, agar plates, conical tubes, culture tubes, antibiotics, burner, toothpicks, incubator, and pipettes. Decontaminate the bench with 70% isopropanol or ethanol to ensure a sterile work environment.

Transfer 10 milliliters of LB media into a 15 milliliter conical tube and add the appropriate antibiotic (carbenicillin or ampicillin based on plasmid resistance). Reflame the tube, cap, and invert to mix the antibiotic thoroughly.

Arrange three sterile culture tubes and label one as negative control and two others with plasmid names (e.g., plasmid A and B). Add your initials and date to each tube, then distribute 2 milliliters of prepared LB-antibiotic media into each culture tube using a sterile serological pipette.

Using a sterile toothpick, select single colonies from your LB agar plate and drop into the labeled culture tubes (avoiding the tube exterior). For the negative control tube, add only a sterile toothpick without any bacterial colony. Replace tube caps after each inoculation.

Place all inoculated culture tubes in a shaking incubator set to the appropriate temperature for your plasmid (found on the Addgene plasmid page). Gentle shaking ensures proper aeration and nutrient availability while preventing bacterial clumping.

After incubation, inspect all tubes for bacterial growth; the negative control should remain clear. If no growth appears, allow more time for culture development, verify antibiotic matches plasmid resistance, confirm colonies were freshly streaked, or increase aeration to improve culture density.

Once adequate culture growth is achieved, spin down the liquid cultures and proceed with plasmid DNA isolation following a standard extraction protocol. Alternatively, create glycerol stocks for long-term storage of your bacterial cultures.