Inoculation is the process by which you introduce bacteria into liquid medium.
In this video we will demonstrate best practices to perform inoculation ensuring sufficient numbers
of bacteria for plasmid DNA isolation. So you may be thinking, "why can't I just grab a chunk of my
glycerol stock in the freezer and throw it in a large volume of media for growth?" As we explained
in a previous video on how to streak bacteria on plates, while the majority of bacterial cells
and a glycerol stock or stab should be genetically identical, there may be an occasional mutant hidden
within the population. By starting a small liquid culture from a single colony, you can ensure that
the liquid culture contains a monoclonal population of cells. Before we begin we'll
need to gather the necessary materials. You will need a spray bottle containing 70% isopropanol
or 70% ethanol, pre-made liquid LB media, LB agar plates containing single colonies of your plasmid
of interest, sterile 15 milliliter conical tubes, sterile culture tubes, appropriate antibiotic for
your plasmid growth, bunson or alcohol burner, autoclave toothpicks or sterile pipette tips,
shaking incubator set at a temperature appropriate for your plasmid, a 10 milliliter serological
pipette, and a pipette aid.
For any work performed at the bench we recommend decontaminating your
bench by wiping down with 70% isopropanol or ethanol. Now you can set up the materials
needed for inoculation. First you will need to prepare your liquid growth medium. We usually use
Luria broth, or LB, as it is the most widely used medium for bacterial culture. If your media does
not contain antibiotics yet, you will need to add the appropriate antibiotic before proceeding. For
any plasmid requested from Addgene, the antibiotic resistance will be listed on the plasma page under
the growth in bacteria section.
Our plasmid contains ampicillin resistance.
At Addgene, our lab used carbenicillin instead of ampicillin.
Carbenicillin is more stable than ampicillin, so it's
more effective at isolating bacteria containing the plasmid of interest.
Always make sure to look up the correct antibiotic concentration needed for plates and
media. You can find a handy table to access this information at addgene.org
Since we will need 10 milliliters of media for our inoculations, we will first transfer 10 milliliters of LB
media to a 15 milliliter conical tube. Now we are ready to add the antibiotics
Add the appropriate amount of antibiotics to the media making sure that the pipette tip does not touch the neck of
the bottle. Reflame the tube, cap with lid, and invert to mix in the antibiotic. Next, arrange 3
culture tubes in a rack. Label the first tube as a negative control, and label the other two tubes
with the name of the plasmid. It's always a good idea to select more than one single colony for
inoculations. To track these samples we will label the tubes plasmid A and plasmid B,
indicating same plasmid, but different colonies. It is also a good idea to add your initials and the date to the tubes.
Transfer 2 milliliters of the LB Carbenicillin media into each culture
tube using a serological pipette. Using a sterile toothpick select the single colony from your LB
agar plate, remove the culture tube cap and drop the toothpick into the culture tube labeled with
your plasmid name and replace the cap. Make sure the toothpick does not touch the outside of the
tube for the negative control tube you will want to simply drop in a sterile toothpick
If you are using an Addgene plasmid, the growth
temperature will be listed under the growth in bacteria section of the plasmid page.
Bacterial liquid culture should be incubated on a gentle shaker in order to ensure proper aeration and
nutrient availability. Incubation using a shaker also avoids bacteria clumping at the bottom of the tubes
To determine if your Addgene plasmid is high or low copy, see the copy number information under the
growth in bacteria section of the plasmid page. After incubation, check all tubes for growth. Your
negative control tube should appear clear with no bacterial growth. So, what happens if, after growing
your culture overnight, you do not observe any bacterial growth? First, allow the culture more
time to grow, as some bacterial cultures have slower growth rates.
Double-check that the antibiotic in your LB media matches the antibiotic resistance for your plasmid. Using the
wrong antibiotic will yield no bacterial growth. If the single colony used for inoculation was
not freshly streaked, you should restreak your bacteria onto a new LB agar plate before growing
in liquid culture. Fresh colonies usually yield greater bacterial growth. More aeration may help
to increase the density of the culture.
Once you obtain adequate culture, growth
you can spin down your liquid cultures and isolate your plasmid DNA by following a
DNA isolation protocol. For long term storage, you can proceed with creating a glycerol stock.
Thank you for watching our inoculation protocol video. Check out our other protocol
pages, including purifying your plasmid DNA, and creating a glycerol stock at
addgene.org/protocols leave a comment below to let us know what videos you'd like to
see in the future, or to tell us how we can improve
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