Antibiotics

2026-06-03

Final concentrations generally recommended.

100 µg/mL Ampicillin (E. coli)
12.5 µg/mL Tetracycline (E. coli)
50 µg/ml Kanamycin (E. coli)
100 µg/mL G418 (S. cerevisiae)
250 µg/mL Hygromycin B (S. cerevisiae)
100 μg/ml clonNAT (S. cerevisiae)
34 µg/ml Chloramphenicol (E. coli for selection)

IMPORTANT

Note the difference between mg and µg. 1 mg = 1000 µg

Ampicillin stock solution (100 mg/ml = 1000 X concentrated)

How to prepare Protocol for ampicillin 100 mg/ml stock solution stock (x 1000).

Weigh ~1 g ampicillin sodium sulfate powder (Fisher 1760-25) in a Falcon 50 mL tube.
Add water to the 10 mL mark and vortex. Adjust the volume with water if necessary.
Filter through 0.22 µm membrane with a new syringe.
Store at -20 in 1 ml aliquots.

Final concentration for E. coli should be 100 µg/ml. This is close to the upper limit recommended by ATCC (see below). The stock solution is stored in 1 mL aliquots marked with a capital “A” at -20°C in the box marked “Antibiotics” in the -20 freezer.

Kanamycin stock solution (50 mg/ml = 1000 X concentrated)

Weigh ~1 g 1 gram of Kanamycin sulfate in a Falcon 50 mL tube.
Add water to the 20 mL mark and vortex. Adjust the volume with water if necessary.
Filter through 0.22 µm membrane with a new syringe.
Store at -20 in 1 ml aliquots.

Final concentration for E. coli should be 50 µg/ml. This is the upper limit recommended by ATCC (see below).The stock solution is stored in 1 mL aliquots marked with a capital “K” at -20°C in the box marked “Antibiotics” in the -20 freezer.

Geneticin (G418) stock solution (200 mg/ml = 1000 X concentrated)

Geneticin (Gibco’s brand name) or G418 (generic name) is used to select for yeast that have the KanMX resistance marker.

Weigh ~2g G418 powder in a Falcon 50 mL tube.
Add water to the 10 mL mark and vortex. Adjust the volume with water if necessary.
Filter through 0.22 µm membrane with a new syringe.
Store at -20 in 1 ml aliquots.

Final concentration for E. coli should be 200 µg/ml. This is less than half of the lower limit concentration recommended by ATCC (see below). The concentration was recommended by Goldstein & McCusker 1999 The stock solution is stored in 1 mL aliquots marked with a capital “G” at -20°C in the box marked “Antibiotics” in the -20 freezer.

Tetracycline stock solution (12.5 mg/ml = 1000 X concentrated)

Add 125 mg Tetracycline Hydrochloride (MW = 480.898 g/mol) powder to a 15 mL FALCON tube.
Add 7 mL of Ethanol 99.5% or 96%.
Add water to 10 mL.
Aliquot 1 mL per tube and store at -20°C.

It is not necessary to sterile filter this solution since it is made in 70% Ethanol. For E. coli XL1-Blue, use 12.5 µg/mL Tetracycline. The stock solution is stored in 1 mL aliquots marked with a capital “T” at -20°C in the box marked “Antibiotics” in the -20 freezer.

Hygromycin B stock solution (250 mg/ml = 1000 X concentrated)

Standard concentration for selection in S. cerevisiae is 300 µg/ml, we use 250 µg/ml. Stock solution is 250 mg/mL (x 1000):

Weigh 2.5 g Hygromycin B powder in a Falcon 15 mL tube.
Add water to the 10 mL mark and vortex. Add more water if necessary.
Filter through 0.22 µM membrane with a new syringe.
Store at -20 in 1 ml aliquots.

The 300 µg/ml selection concentration was recommended by Goldstein & McCusker 1999. The stock solution is stored in ~1 mL aliquots marked with a capital “H” at -20°C in the box marked “Antibiotics” in the -20 freezer.

IMPORTANT

Hygromycin might be necessary in larger concentrations than stated here as there might be strain variability.

Nourseothricin (clonNAT) stock solution (100 mg/ml = 1000 X concentrated)

Nourseothricin is a mixture of Streptothricins C, D, E and F and can be used as selection antibiotic for a broad spectrum of pro- and eukaryotic organisms (i.e. Gram-positive and Gram-negative bacteria, yeast, filamentous fungi, protozoa, micro algae, plants and more.

Weigh 1 g clonNAT powder in a Falcon 15 mL tube.
Add water to the 10 mL mark and vortex. Add more water if necessary.
Filter through 0.22 µM membrane with a new syringe.
Store at -20 in 1 ml aliquots.

The concentration was recommended by Goldstein & McCusker 1999 The stock solution is stored in 1 mL aliquots marked with a capital “N” at -20°C in the box marked “Antibiotics” in the -20 freezer.

Werner Bioagents recommended:

Organism	conc (µg/mL)
Escherichia coli	50
Saccharomyces cerevisiae	100
Ustilago maydis	75
Leishmania sp.	>100
Cryptococcus neoformans	100
Arabidopsis thaliana	100

Chloramphenicol stock solution (34 mg/ml = 1000 X concentrated)

To make a 34 mg/mL chloramphenicol stock solution you must dissolve chloramphenicol powder in 100% or 95% ethanol, as it is poorly soluble in water.

Component	Amount
Chloramphenicol Powder	340 mg
Ethanol (100% or 95%)	10 mL

Weigh 340 mg of chloramphenicol powder using an analytical balance and transfer it into a sterile 15 mL conical tube (such as a Falcon tube).
Add the solvent: Measure 9 mL of 100% ethanol and add it to the tube first.
Dissolve completely: Cap the tube tightly and vortex or invert it vigorously until the powder is fully dissolved and the liquid is completely clear.
Bring to volume: Top off the liquid level to exactly the 10 mL mark with ethanol.
Divide the solution into 1 mL aliquots in sterile microcentrifuge tubes. Label and store at -20°C.

There is no need to filter sterilize this solution. Because it is made in high-concentration ethanol, the solution is sterile.

Standard Usage (1000X Concentration)

When adding this to growth media (like LB broth or LB agar), use a 1:1000 dilution. For example, add 1 mL of stock solution to 1 L of cooled media, resulting in a final working concentration of 34 µg/mL. Always let agar cool to approximately 50°C–55°C before adding the antibiotic to prevent heat degradation. [1, 10, 11, 12]

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