Jan 01, 2020
Inoculating a Liquid Bacterial Culture
- 1Addgene
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2020. Inoculating a Liquid Bacterial Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.5abg2an
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: July 09, 2019
Last Modified: August 18, 2020
Protocol Integer ID: 25635
Abstract
This protocol is for inoculating a liquid bacterial culture. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/inoculate-bacterial-culture/.
Guidelines
Tips and FAQ
- What is the difference between high copy and low copy plasmids? The copy number refers to the number of copies of an individual plasmid within a single bacterial cell. Large plasmids usually have a low copy number (approximately one or two copies per cell) and they need to grow for longer periods of time (approximately 18:00:00 -30:00:00 ). On the other hand, smaller plasmids can be present in large numbers, 50 or more per cell and have a high copy number. High copy number plasmids should only need to be grown for 12:00:00 -16:00:00 on average. Certain features of a plasmid may render it low copy regardless of plasmid size. See the plasmid's info page to determine if your plasmid is high or low copy.
- I didn't get any growth after overnight incubation. What went wrong? Try growing the culture for more time. Some bacterial cultures grow more slowly. Also, bacteria incubated at 30 °C rather than 37 °C often require longer incubation times. Double-check that the antibiotic in your LB media matches the antibiotic resistance on your plasmid. If the bacteria on your LB agar plates are not fresh, you should streak your bacteria onto a new LB agar plate before growing in liquid culture. More aeration may help to increase the density of the culture. Normally cultures shake at 150 - 250 rpm, increase this to 350 - 400 rpm to obtain a higher cell density.
Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle:
- 4 g NaCl
- 4 g Tryptone
- 2 g Yeast Extract
- add dH2O to 400 mL
Note
Note, if your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.
Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!) and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature. Now screw on the top of the bottle and store the LB at Room temperature .
When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration (see table below in step 9).
Note
Note, if you intend to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2 L Erlenmeyer flask.
Drop the tip or toothpick into the liquid LB + antibiotic and swirl your tube or flask.
Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
Incubate bacterial culture at 37 °C for 12:00:00 -18:00:00 in a shaking incubator.
Note
Note, some plasmids or strains require growth at 30 °C . If so, you will likely need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at lower temperatures.
After incubation, check for growth, which is characterized by a cloudy haze in the media (see image below).
Figure 1: Media without growth (top) and with growth (bottom)
Note
Notes:
- Some protocols require bacteria to be in the log phase of growth. Check the instructions for your specific protocol and conduct an OD600 to measure the density of your culture if needed.
- A good negative control is LB media + antibiotic without any bacteria inoculated. You should see no growth in this culture after overnight incubation.