UA Biophysics:Protocols:Ampicillin

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Ampicillin sodium salt

Ampicillin is a ß-lactam antibiotic that inhibits bacterial cell-wall synthesis by inactivating transpeptidases on the inner surface of the bacterial cell membrane. Ampicillin in solution is a function of pH, temperature and the identity of the buffer. It’s activity is quickly lost when stored above pH 7.


Ampicillin is usually used at a concentration of 50 - 100 µg/mL. Stock solution is typically at 25mg/mL


  1. According to desired amount of antibiotics, calculate the amount in grams that has to be weighed.
    1. For example, for 10mL of 25mg/mL of Ampicillin:


  1. Ampicillin is soluble in ddH2O.
  2. Filter sterilize solution using a 0.2 µm syringe filter.
  3. Divide in aliquots of 500μL
  4. Store at -20 °C. up to 4-6 months

Optimal storage conditions are suggested as 2-8˚C, and pH 3.8-5 where its activity was retained at 90%+ for a week. At 37°C in culture, ampicillin is stable up to 3 days.


For culture plates, allow media to cool to 55 °C before adding ampicillin to a final concentration of 35-50 μg/ml.

For Example, for 300 mL of media at a final concentration of 50µg/mL:

Ci Vi = Cf Vf

25mg/mL* Vi = 0,05mg/mL*300mL

Vi = 15mg/(25mg/mL)

Vi = 0,6 mL

Please, Do not hesitate to contact me if you have any question Lina Contreras

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