How to Make Stock Solutions for Antibiotics and Cell Selection Agents

by Simon Currie

Antibiotics and cell selection agents are used to isolate cells that contain a particular resistance marker from a mixed population. These powerful reagents are used in standard molecular cloning, protein expression, genetic screens, establishing stable cell lines, and more.

Making stock solutions for antibiotics and cell selection agents involves weighing out the powder, solubilizing it in deionized water or ethanol, filter sterilizing the solution, and storing it in single-use aliquots. GoldBio’s EZ pak^TM formats take the guess work out of these steps and makes stock solution preparation a breeze.

While antibiotics and cell selection agents technically have slightly different meanings, for the purpose of this article (making stock solutions) these terms are completely interchangeable. For brevity I’ll just use the term “antibiotic” throughout, but you would follow the same exact protocol for cell selection agents.

Step-by-step antibiotic stock solution instructions

1. Weigh out the amount of antibiotic you’re going to use based on the desired volume and concentration of stock solution. For example, in Figure 1 we show a calculation for making 100 mL of 100 mg/mL antibiotic stock solution. 
2. Pour the weighed antibiotic into a conical tube or a graduated cylinder and add deionized water up to the desired volume.
   1. Note that most antibiotics and cell selection agents are soluble in water. A few, such as chloramphenicol and tetracycline have poor water solubility and stock solutions should be made using 100% Ethanol. When later using the stock solution, the ethanol will be effectively diluted out as long as your stock concentration is high enough (at least 100x or more compared to the working concentration).
3. Solubilize the antibiotic. If using a conical tube, cap it and invert several times until the antibiotic goes into solution. If using a graduated cylinder, add a stir bar and allow the solution to stir until the antibiotic is solubilized.
4. After the antibiotic is in solution, re-check the volume and top up to the intended volume.
5. Filter the stock solution with a syringe filter or a bottle-top filter. This is important for sterilizing the antibiotic solution to make sure that you don’t contaminate your cultures.
   1. Sterilizing your antibiotic solution is an important step for water-based stock solutions. If you’re antibiotic stock solution is in 100% ethanol you really don’t need to filter sterilize it, though it doesn’t hurt either. Do not heat sterilize any antibiotic solution as this will degrade the antibiotics.
6. Pipet the solution into convenient single-use volumes.
7. Store single-use aliquots in the -20 °C freezer until they are ready for use.  

Figure 1. Example calculation for how much antibiotic you need to weigh out to make 100 mL of a 100 mg/mL stock solution. The middle step is simply the conversion between grams and milligrams.  

That’s all there is to making antibiotic stock solutions. When stored at -20 °C and used without repeated freeze-thaw cycles, these aliquots are good for at least 3-6 months. If you’re unsure if your older stock solutions are still potent, simply test them to see.

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antibiotics Cell Selection Simon Currie

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