Antibiotic Stock Solution Mixing
Standard Antibiotic Stock (and Working) Concentrations:
Ampicillin: 100 mg/ml
Working Concentration: 100 µg/mL
Chloramphenicol: 25 mg/ml (usually) or 12.5 mg/ml (for fosmids)
Working Concentration: 25/12.5 µg/mL
Kanamycin: 50 mg/ml
Working Concentration: 50 µg/mL
Streptomycin: 200 mg/ml
Working Concentration: 200 µg/mL
Gentamicin: 10 mg/ml
Working Concentration: 10 µg/mL
Carbenicillin: 100 mg/ml
Working Concentration: 100 µg/mL
Hygromycin B: 200 mg/ml
Working Concentration: 200 µg/mL
Tetracycline: 10 mg/ml (light-sensitive! wrap plates/broths in foil)
Working Concentration: 10 µg/mL
Calculating Working Concentration
These stock solutions are x1000 concentration, which is extremely handy for diluting into media. When you take out your bottle of premade antibiotic stock, you can determine how much to use by looking at the volume of media you plan to add it to. This equation does not vary between antibiotics, as long as you mix them to the correct concentration.
Volume of stock to add = (total volume of media) / 1000
Example 1: You have 15 ml of LB Media and want to add Ampicillin. You should add 15 µl of Ampicillin stock solution to the media.
Example 2: You have prepared 400ml of LB Agar media and want to add Chloramphenicol. You should add 400 µl of Chloramphenicol stock solution to the media.
Equipment & Consumables:
PPE: Gloves, Lab Coat and Goggles
You should be aware of any antibiotic allergies as well.
Antibiotic powder will have an adverse reaction for some individuals.
Sterile microcentrifuge/eppendorf/eppi tubes or 15 ml falcon tubes
Weighing Scale (accurate to to 0.001g)
Weigh boats (or folded paper)
Powder spatula or a teeny-tiny spoon.
If you have an inaccurate scale, you may have to mix a larger volume of stock solution in order to make up for the lack of precision.
Antibiotic powder with which you wish to make a stock solution of e.g. Ampicillin powder
Sterile RO/MilliQ Water (dH2O)
Most antibiotic stock should be made by adding the powder directly to sterile water. Use water unless otherwise indicated.
95% Ethanol
Chloramphenicol powder should be dissolved in 95% Ethanol instead of water for maximum storage potential
Syringe
0.22 µm syringe filter
Protocol:
Recipe is for 1 ml of Stock Solution.
x10 all values for 10 ml of stock solution.
Use the above list of recommended stock concentrations to determine how many milligrams of powder you need to dissolve in 1 ml of liquid.
e.g. For ampicillin, we need to dissolve 100 mg of powder in 1 ml
e.g. For chloramphenicol, we need to dissolve 25 mg of powder in 1 ml
Zero your scale with the weigh boat on it and then weigh out that amount of antibiotic powder onto your weight boat.
Set up an eppendorf tube and add 1 ml of dH2O (or Ethanol if you’re dissolving chloramphenicol)
Gently tip your powder into the tube of liquid, using a spatula to ensure every last crumb ends up in the tube.
Close the lid and use the vortex to dissolve the powder. This should take less than 60 seconds, but can sometimes be slow.
Filter sterilise through the syringe filter.
Freeze at -20°C.
Relatively stable across many freeze/thaw cycles
Ampicillin is far less stable than the other antibiotics, especially once it is in liquid or agar. Keep this in mind. I’d happily mix 10 ml of Chloramphenicol stock for the year, but I’d rather mix Ampicillin Stock every 2-3 months.