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1: Inoculation

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  • Page ID
    79490
    • Nathan Reyna, Ruth Plymale, & Kristen Johnson
    • Ouachita Babtist University & University of New Hampshire

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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.

    1. You will use a sterile toothpick to pick the colony, and you can simply drop the entire toothpick into the liquid culture tube.
    2. 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).
    3. 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

    This page titled 1: Inoculation is shared under a not declared license and was authored, remixed, and/or curated by Nathan Reyna, Ruth Plymale, & Kristen Johnson.

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