Benzylpenicillin Assay

Reagents and Materials

  • 96-well plate
  • 0.5% m/v Phenol Red
  • Benzylpenicillin
  • NaOH 1M
  • Purified Beta-lactamase 1µg/µL
  • pH strip


  1. Add different amounts Beta-lactamase to the wells (we added 5µg to 25µg of purified TALE A-linker-beta-lactamase)
  2. To a tube add 16.6mL of ddH2O (deionized water), 2mL of 0.5% m/v Phenol Red and 1.2g of benzylpenicillin. Add the pH to 8.5 with NaOH 1M
  3. Add 100µL of the solution above to each well
  4. Incubate at room temperature for 10 minutes
  5. Observe color change from red to yellow
  6. Measure pH with a pH strip (the pH is expected to drop)