Mar 12, 2026
Preparing an Overnight Culture
This protocol is a draft, published without a DOI.
- Tamra Lahom1
- 1Carleton College
Protocol Citation: Tamra Lahom 2026. Preparing an Overnight Culture. protocols.io https://protocols.io/view/preparing-an-overnight-culture-jv2jcn8cp
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 12, 2026
Last Modified: March 12, 2026
Protocol Integer ID: 313131
Keywords: culture bacteria in the lab, overnight culture of liquid lb, overnight culture plasmid, culture bacteria, enough plasmid dna for experimental use, liquid culture, specific plasmid, plasmid, bacteria, enough plasmid dna, colonies of bacteria, bacteria in the presence, more antibiotic resistance gene, antibiotic resistance gene, sufficient numbers of bacteria, luria broth, antibiotic, higher density of bacteria, liquid lb, specific antibiotic, lab, lb agar plate, overnight culture, experimental use
Abstract
Plasmids can carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily isolate bacteria containing that plasmid from bacteria that do not contain it by artificial selection (i.e., growing the bacteria in the presence of the antibiotic).
Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal) colonies of bacteria carrying a specific plasmid. However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers of bacteria necessary to isolate enough plasmid DNA for experimental use. The following protocol is for inoculating an overnight culture of liquid LB with bacteria.
Troubleshooting
Prepping Culture
Obtain a 14 mL culture tube
Add 5 mL of LB media.
Add 5 µL of antibiotic stock.
Using a sterile pipette tip, select a single colony from your LB agar plate or a glycerol stock.
Note
The glycerol stock should never thaw. Keep on ice but in the bubble of sterility.
Drop the tip into the liquid LB + antibiotic and swirl.
Loosely cover the culture with sterile aluminum foil or the cap that is not air tight.
Incubate bacterial culture at 37 °C for 12–18 h in a shaking incubator