AMPs efficiency and MIC assay

Reagents:

1. Staphylococcus aureus solution (37℃, 200rpm, overnight culture);
2. Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China Peptides, Suzhou); LB agar plates

Procedure:

• AMPs dilution
1. Take AMPs stock solution from -20℃ freezer.
2. Dilute AMPs by PB buffer. The conc. gradients of diluted AMPs as follow:
   LL-37: 80ug/ml, 40ug/ml, 20ug/ml, 10ug/ml, 5ug/ml, 1ug/ml, 0.5ug/ml;
   Grammistin-Pp1: 40ug/ml, 20ug/ml, 10ug/ml, 6ug/ml, 3ug/ml, 1ug/ml, 0.5ug/ml
   GF-17: 20ug/ml, 10ug/ml, 6ug/ml, 3ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml
3. After each dilution, vortex 1-3min for each of concentration of AMPs
• Staphylococcus aureus dilution
1. Measure OD₆₀₀ value of original Staphylococcus aureus solution.
2. Dilute Staphylococcus aureus solution 500 times and 1000 times respectively in different EP tube by PB buffer;
3. After dilution by PB buffer, vortex each conc. gradients of solution 1-3min.
• AMPs and Staphylococcus aureus interaction
1. Pipette 50μl diluted AMPs solution and 50μl diluted Staphylococcus aureus solution (1/500 and 1/1000 diluted) into one EP tube.
2. Vortex each EP tube for 1-3 min.
3. Incubate 3.5 hours at 37℃, 180rpm condition.
4. Plate 30μl mixed solution into the LB agar medium. Overnight culture at 37℃ condition.