Team:LCG-UNAM-Mexico/Resources/Protocols/Titering Protcol

TITERING PROTOCOL
To titer phage, 0.25 ml aliquots of a fresh culture of bacteria are added to a series of 13 × 100 mm tubes at room temperature. Appropriate dilutions of phage in 100 μl buffer are added to the cells, followed by the addition of 2.5–3 ml of melted top agarose (plus IPTG for 5615 strains) at 45–50°C. The contents of the tube are immediately mixed and then poured in a standard 100 × 15 mm Petri dish containing 20 ml of hardened TB or LB agar. Wild-type plaques will appear within 2–3 h at 37°C, as soon as the lawn becomes turbid enough to see them; some recombinants may take longer to develop. In most cases, plates should not be incubated overnight at 37°C because the plaques become too large; however, plaques will usually remain a manageable size if plates are left overnight at room temperature. For additional details regarding phage titering by plaque assay, refer to Plaque Assay below.