Can I Put Bacteria at 4 C Then Grow Them Again

Inoculating a Liquid Bacterial Culture

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Background Information

Plasmids can bear one or more antibody resistance genes, which confer resistance to a specific antibody to the bacteria conveying them. The presence of an antibiotic resistance factor on a plasmids allows researchers to easily isolate bacteria containing that plasmid from bacteria that do not contain it by artificial selection (i.e. growing the leaner in the presence of the antibiotic).

Luria broth (LB) is a nutrient-rich media ordinarily used to culture leaner in the lab. LB agar plates are frequently used to isolate individual (clonal) colonies of leaner carrying a specific plasmid. Notwithstanding, 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 employ. The following protocol is for inoculating an overnight culture of liquid LB with bacteria.

Video

Watch the protocol video beneath to learn how to inoculate bacteria in liquid culture.

Protocol

1. Set liquid LB. For case, to make 400 mL of LB, weigh out the following into a 500 mL drinking glass canteen:

   • 4 m NaCl
   • four one thousand Tryptone
   • two g Yeast Extract

   • and dH₂O to 400 mL

   Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients to a higher place.

   Media without growth (tiptop) and with growth (bottom)

   

   

   Loosely close the cap on the canteen (do Non close all the way or the bottle may explode!) then loosely cover the entire tiptop of the bottle with aluminum foil. Autoclave and allow to cool to room temperature. Now spiral on the meridian of the bottle and store the LB at room temperature.

2. When prepare to grow your civilization, add liquid LB to a tube or flask and add the appropriate antibody to the right concentration (see table below).

   Note: If yous intend to do a mini-prep you volition usually desire to start 2 mL in a falcon tube, but for larger preps you might want to utilise as much as a liter of LB in a 2 50 Erlenmeyer flask.

3. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.

4. Drop the tip or toothpick into the liquid LB + antibody and swirl.

5. Loosely cover the civilization with sterile aluminum foil or a cap that is not air tight.

6. Incubate bacterial civilization at 37°C for 12-18 60 minutes in a shaking incubator.

   Note: Some plasmids or strains require growth at 30°C. If then, you volition probable need to grow for a longer time to become the correct density of bacteria since they volition grow more than slowly at lower temperatures.

7. Later on incubation, cheque for growth, which is characterized past a cloudy brume in the media (see right).

   Note: 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 civilization if needed.

   Note: A good negative command is LB media + antibiotic without whatever bacteria inoculated. You should see no growth in this civilization after overnight incubation.

8. (Optional) For long term storage of the leaner, you can go on with Creating a Glycerol Stock.

9. Yous can now isolate your plasmid DNA from the bacterial civilization by post-obit Isolating Your Plasmid Dna.

Antibiotic Concentrations

Ordinarily Used Antibiotics	Recommended Concentration
Ampicillin	100 µg/mL
Bleocin	5 µg/mL
Carbenicillin	100 µg/mL
Chloramphenicol	25 µg/mL
Coumermycin	25 µg/mL
Gentamycin	10 µg/mL
Kanamycin	50 µg/mL
Spectinomycin	fifty µg/mL
Tetracycline	10 µg/mL

Tips and FAQ

• What is the departure between high copy and low copy plasmids?

  The copy number refers to the number of copies of an private plasmid within a single bacterial cell. Large plasmids usually have a low re-create number (approximately one or two copies per cell) and they need to grow for longer periods of fourth dimension (approximately eighteen-30 hr). On the other hand, smaller plasmids tin can be present in large numbers, 50 or more than per prison cell and have a high re-create number. High copy number plasmids should merely need to be grown for 12-16 hr on average. Certain features of a plasmid may render it depression copy regardless of plasmid size. Run across the plasmid's info page to determine if your plasmid is high or low copy.

• I didn't get whatever growth afterwards overnight incubation. What went wrong?

  Try growing the civilisation for more than time. Some bacterial cultures grow more slowly. Also, bacteria incubated at 30°C rather than 37°C often crave 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 lot should streak your bacteria onto a new LB agar plate before growing in liquid civilisation.

  More than aeration may help to increase the density of the civilization. Normally cultures shake at 150 - 250 rpm, increase this to 350 - 400 rpm to obtain a higher cell density.

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Source: https://www.addgene.org/protocols/inoculate-bacterial-culture/

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