Team:XJTLU-CHINA/Protocols AMPs efficiency MIC assay
AMPs efficiency and MIC assay
Reagents:
- Staphylococcus aureus solution (37℃, 200rpm, overnight culture);
- Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China Peptides, Suzhou); LB agar plates
Procedure:
- AMPs dilution
- Take AMPs stock solution from -20℃ freezer.
- 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 - After each dilution, vortex 1-3min for each of concentration of AMPs
- Staphylococcus aureus dilution
- Measure OD600 value of original Staphylococcus aureus solution.
- Dilute Staphylococcus aureus solution 500 times and 1000 times respectively in different EP tube by PB buffer;
- After dilution by PB buffer, vortex each conc. gradients of solution 1-3min.
- AMPs and Staphylococcus aureus interaction
- Pipette 50μl diluted AMPs solution and 50μl diluted Staphylococcus aureus solution (1/500 and 1/1000 diluted) into one EP tube.
- Vortex each EP tube for 1-3 min.
- Incubate 3.5 hours at 37℃, 180rpm condition.
- Plate 30μl mixed solution into the LB agar medium. Overnight culture at 37℃ condition.
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