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Team:MIT/Tooth binding assay protocol
From 2008.igem.org
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Tooth binding assay
• Wash 50 mg HA beads 3 times with 1mM phosphate buffer (PB) and equilibrate in PB for 2 hours
• HA beads settle for 30 to 60 s, then remove supernatant
• Control
o add 1mM phosphate-phosphate buffered saline (PBS) for 1h
o wash with 10mM PBS
o add saliva for 1h
o wash with 10mM PBS
• Variable
o Add saliva (amount?) for 1h
o Wash with 10mM PBS
o Add p1025 (amount?) extract
o Wash with 10mM PBS
• Add 1mL S. Mutans suspension (1-2*10^7 colony-forming units (CFU))
• Extract 100 μL supernatant after 5 minutes and plate onto Todd Hewitt Broth (THB), or whatever media the S. Mutans comes in
• Repeat above after 1h, 2h
• Count colonies (approximately CFUs) on the three plates and calculate the amount attached to HA beads through the relation “CFU supernatant time 0 – CFU supernatant time 1h, 2h = CFU on HA beads"
PBS—naCL 8.0 g L-1, KCl 2.0 g L-1, Na2HPO4, 2H20 2.0 g L-1, KH2PO4 2.0 g L-1; pH 7.2
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