Jun 11, 2026

Overnight Bacterial Cultures

DOI
https://dx.doi.org/10.17504/protocols.io.14egn5r5zg5d/v1
  • Raven Coffer1,
  • Owen Butler1
  • 1DAMP Lab
  • DAMP Lab
DAMP Lab
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn5r5zg5d/v1
Protocol Citation: Raven Coffer, Owen Butler 2026. Overnight Bacterial Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn5r5zg5d/v1
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 25, 2026
Last Modified: June 11, 2026
Protocol  Integer ID: 313875
Keywords: overnight bacterial culture, bacterial culture, including dna extraction, protein purification, dna extraction, glycerol stock
Abstract
The following protocol provides instruction on how to create bacterial cultures starting from glycerol stocks or agar plates. Bacterial cultures are useful for downstream applications including DNA extraction, protein purification, or otherassays.
Guidelines
  • Inoculation loops: Only touch the top third (furthest from the tip) of the inoculation loop. This will help prevent contamination of the culture.
  • Picking colonies: Always pick single colonies from a plate. Colonies that touch or are close to others may not have the desired plasmid.
  • Glycerol stocks: Be extremely careful when handling glycerol stocks. Minimize exposure (always close lid when not in use). Always keep on DRY ICE. Return to the -80 freezer immediately when finished inoculating.
  • Logical ordering: Inoculate culture tubes in a logical order, to keep track of the step you are on. Additionally, when a culture tube has already been inoculated, move it to a different physical location (a new tube rack) so you can track completed vs incomplete.
  • Aseptic Technique: Perform all work close to the bunsen burner.
Materials
Consumables
  • 15 mL snap-cap culture tubes
  • Sterile loops
  • Filtered pipette tips
  • Dry Ice (if using glycerol stocks)

Equipment
  • Bunsen burner
  • Pipettes
  • Serological pipette
  • 250 mL erlenmeyer flask (sterile)

Reagents
  • Appropriate antibiotic (ampicillin, kanamycin, etc.)
  • Appropriate liquid culture media (LB, M9, etc.)

Before start
Know the growth conditions (optimal incubation time, speed, and temperature) of your samples.
  • For E. coli, DAMP Lab recommends incubation at 275 rpm, 37°C for 16 -18 hours

Preparation
7m 30s
Decontamination
Wipe benches and pipettes with DNAaway, RNase Aaway. Afterwards wipe benches and pipettes with 70% ethanol.
2m
Setup
Light the bunsen burner. All work should be performed close to the bunsen burner.
30s
Label
Label culture tubes with the name of the construct (or shortened nickname), your initials, and the date.
Note
If you are using nicknames, you must create a table in your ELN detailing the designated nickname for each construct. See example below:

Nickname: KZK10_A
Full name: DHY213_KZK10_02302030_A

5m
Procedure
13m 10s
Sample Preparation
To the erlenmeyer flask, add the desired amount of culture media. Use the following formula to calculate total culture media where x = number of cultures:
(5 mL * x) + 2 mL = total culture media required
3m
To erlenmeyer flask, add the antibiotic at a 1:1000 ratio.

Note
For example: For 17 mL of media, add 17 µL of ampicillin.

2m
Swirl flask for approximately 00:00:10 seconds to fully mix.
10s
Using a serological pipette, aliquot 5 mL culture media + antibiotic to each labeled culture tube.
2m
Sample Preparation
Step case

Glycerol Stocks
9 steps

Obtain the appropriate glycerol stock from storage at -80 °C on DRY ICE.

Use an inoculation loop to gently scrape the top of the frozen glycerol stock. Once you observe a small amount of opaque liquid on the tip of the loop, dip into the corresponding culture tube and swirl for 00:00:10 seconds.

Discard the loop into the sharps container. Repeat for each sample.

Note
  • Be extremely careful when handling glycerol stocks. Minimize exposure (always close lid when not in use). Always keep on DRY ICE.
  • Only touch the top third (furthest from the tip) of the inoculation loop. This will help prevent contamination of the culture.

5m
Storage
Return glycerol stocks to storage at -80 °C immediately after use.
Incubation
Place the culture tubes in the incubator tube rack at the appropriate temperature for the sample. Ensure the lids are moveable (not locked down) so that air can flow into the tubes.
1m
Incubate the cultures for the recommended duration at the appropriate shaking speed for the sample.
Cleanup
3m
Decontamination
Rinse erlenmeyer flask with water in the sink, hang on the drying rack to be washed.
1m
Wipe benches and pipettes with DNAaway, RNase Aaway. Afterwards wipe benches and pipettes with 70% ethanol.
2m
Acknowledgements
Drafted by: Owen Butler
Uploaded by: Gabriella Kong
Edited by: Raven Coffer, Owen Butler, Kristen Sheldon, Lena Landaverde, Molly Brennan