1.7: Inoculation
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Inoculation of Overnight Cultures
Small sample of bacteria is taken from a plate and placed in LB broth to be used in ZymoPure miniprep or for long term storage.
| Materials | Storage Temperature |
|---|---|
| Sterile Toothpicks | Room Temp |
| LB Broth | Room Temp |
| Isolated Colony of Transformed Bacteria | Room Temp |
Procedure:
Using sterile technique, pick an isolated colony from a fresh plate (less than seven days old) and inoculate LB-Miller medium.
- You will use a sterile toothpick to pick the colony, and you can simply drop the entire toothpick into the liquid culture tube.
- Sterile 5 mL of LB broth will be provided for you for this step. You may add the required antibiotic to the LB prior to inoculating with your colony (optional).
- Incubate with shaking for 8–16 hours at 37°C before harvesting. This results in maximum yields of a high-copy-number plasmid.
(Typically, after overnight incubation, the absorbance of a tenfold dilution of the culture at a wavelength of 600nm (A600) with a 1cm path length should range from 0.10–0.35.)
These cultures can now be used in the Mini-prep procedure or stored long term in glycerol. (See Appendix VI for how to make your own glycerol stock.)
Growth of Overnight Cultures – Helpful Hints if you are having difficulty.
Different culture media will also have a profound effect on the growth of different bacterial strains. Most plasmid DNA purification systems are appropriate for bacterial cultures grown in 1X Luria-Bertani (LB) medium. However, use of a LB-Miller medium containing more NaCl will produce significantly greater yields and is highly recommended. Richer media such as 2X YT, CIRCLEGROW® or Terrific Broth may be used to increase plasmid yields by increasing the biomass for a given volume of culture. Keep the biomass in a range acceptable for the plasmid isolation system used, as overloading may result in poor purity and yield of the plasmid DNA
