Working with Plasmids

Bacterial cultivation media and antibiotics

You should always grow your bacterial cultures for plasmid preparation from a single colony picked from a freshly streaked selective plate. Transferring bacteria directly from glycerol stocks, agar stabs, or liquid cultures is not recommended in microbiology as it can result in plasmid loss. Similarly, using plates stored for extended periods may cause plasmid loss or mutation. To isolate single colonies, it's best to streak a desired clone from a glycerol stock onto a freshly prepared agar plate with the suitable selective agent. Repeat the procedure to ensure that a single colony of an antibiotic-resistant clone can be picked.

Liquid cultures of E. coli can generally be grown in LB (Luria-Bertani) medium. Please note, however, that a number of different LB broths with different compositions are commonly used. Different formulations contain different concentrations of NaCl and give rise to varied yields of plasmid DNA. We recommend using the LB composition in the table LB medium to obtain the highest yields of plasmid DNA.

LB medium
Component Amount per liter
Tryptone 10 g
Yeast extract 5 g
NaCl 10 g

For preparing 1 L of LB medium, add 10 g NaCl, 10 g tryptone, and 5 g yeast extract to 950 mL distilled or deionized water and shake or stir until dissolved. Adjust the pH to 7.0 with 5 M NaOH. Adjust the volume of the solution to 1 L with distilled or deionized water. Decant into smaller aliquots and sterilize by autoclaving (see Sterilizing media).

Tip: It is advisable to autoclave liquid medium in several small bottles rather than in one large vessel to avoid possible contamination of an entire batch. After autoclaving, do not use medium for 24 hours to ensure it is properly sterilized and free of contaminating microorganisms.

Tip: Antibiotics should be added to liquid medium immediately before use from stock antibiotic solutions that have been filter-sterilized, distributed into aliquots, and stored in the dark at –20°C (see Antibiotics).

Sterilize liquid or solid media by autoclaving, using a pressure and time period suitable for the type of medium, bottle size, and autoclave type.

Tip: Fill bottles only 3/4 full with medium and loosen the caps before autoclaving to avoid hot medium boiling over. Tighten caps once the medium is cool (<40°C) to keep it completely sterile.

Tip: Antibiotics and nutrients such as amino acids are inactivated by the high temperatures of an autoclave. They should be sterilized by filtration through a filter unit with a pore size of 0.2 µm and added to the cooled, autoclaved medium from properly stored stock solutions.

E. coli strains can generally be streaked and stored on LB plates containing 1.5% agar and the appropriate antibiotic(s).

Preparation: Prepare LB medium according to the composition given in the table above. Just before autoclaving, add 15 g agar per liter and mix. After autoclaving, swirl the medium gently to distribute the melted agar evenly throughout the solution. Take care that the hot liquid does not boil over when swirled.

Tip: Cool autoclaved agar medium to below 50°C (when you can hold it comfortably) before adding heat-sensitive antibiotics and nutrients. Mix thoroughly to obtain an even concentration throughout the medium before pouring.

Tip: Pour plates in a laminar flow hood or, if no hood is available, on a cleaned bench surface next to a Bunsen. Use 30–35 mL medium per standard 90 mm petri dish (~30 plates per liter of medium).

After pouring plates, any air bubbles may be removed by passing the flame of a Bunsen burner briefly over the surface. Do not linger with the flame, as this may destroy antibiotics in sections of the plates.

Dry plates either directly after solidification or just before use by removing the lids and standing the plates in a laminar-flow hood for 1 hour. Alternatively, if you cannot access a hood, plates can be dried with the covers slightly open in a 37°C incubator for 30 min or left upside down with lids on at room temperature for 2–3 days.

Tip: Store plates inverted at 4°C in a dark room or wrapped in aluminum foil to preserve light-sensitive antibiotics. Do not store for longer than 3 months, as antibiotics may degrade. 

Bacterial strains carrying plasmids or genes with antibiotic selection markers should always be cultured in liquid or on solid medium containing the selective agent. Lack of antibiotic selection can lead to loss of the plasmid carrying the genetic marker and potentially to selection of faster-growing mutants!

Tip: Prepare stock solutions of antibiotics separately from batches of liquid or solid media, sterilize by filtration, aliquot, and store in the dark at –20°C. Recommended stock and working concentrations for commonly used antibiotics are shown in the table Concentrations of commonly used antibiotics.

Tip: Before adding antibiotics to freshly autoclaved medium, ensure the medium has cooled to below 50°C.

Concentrations of commonly used antibiotics
Antibiotic Stock solution concentration
 Storage temperature Working concentration (dilution)
Ampicillin (sodium salt) 50 mg/mL in water  –20°C 100 µg/mL (1/500)
Chloramphenicol 30 mg/mL in ethanol –20°C 170 µg/mL (1/200)
Kanamycin 10 mg/mL in water –20°C 50 µg/mL (1/200)
Streptomycin 50 mg/mL in water –20°C 50 µg/mL (1/200)
Tetracycline HCl 5 mg/mL in ethanol –20°C 50 µg/mL (1/100)

Photometric measurements of cell density can vary between different spectrophotometers. The optical density reading of a bacterial culture is a measure of the light scattering, which varies depending on the distance between the sample and the detector. You will need to calibrate each individual spectrophotometer for accurate conversion of OD600 measurements into the number of cells per mL. You can do this by plating serial dilutions of a culture onto LB agar plates in the absence of antibiotics. You can then use the counted colonies to calculate the number of cells per mL, which is then set in relation to the measured OD600 values. 

If you’re unable to get spectrophotometric measurements of the cell density or calibrate your photometer, you can estimate the amount of cells harvested by measuring the wet weight of the pellet. Typically, a 1-liter overnight shaker culture of E. coli with a cell density of 3–4 x 109/mL corresponds to a pellet wet weight of approximately 3 g/L.

a man sitting in an armchair at a table and reading
Carlos: Learning about culture media and antibiotics was fascinating! It's incredible how you can manipulate environments to study bacteria. But what really piqued my interest was the mention of bacterial transformation. I've got to dive deeper into this. How do bacteria actually take up foreign DNA? How do you actually do this?