Anti dig antibody

Strains containing the defined phage, SaPI(s) and/or plasmids were grown to early exponential phase (OD₅₄₀~0.15) in 10 ml of TSB supplemented with antibiotics where plasmids were present. Phages were induced with mitomycin C (2 μg ml^-1) and, where pCN51 expression plasmid derivatives were present, 1-5 μM CdCl₂ was added to induce expression as indicated. One ml samples were taken at the defined timepoints, pelleted by centrifugation (16000 × g, 2 min) and shock frozen on dry ice. The samples were re-suspended in 50 μl lysis buffer (47.5 μl TES-Sucrose (10 mM Tris-Cl, 100 mM NaCl, 1 mM EDTA, 20% (w/v) sucrose) and 2.5 μl lysostaphin [12.5 μg ml^-1]) and incubated at 37°C for 1 h. Following the incubation, 55 μl of SDS 2% proteinase K buffer (47.25 μl H2O, 5.25 μl SDS 20%, 2.5 μl proteinase K [20 mg ml^-1]) was added before incubation at 55°C for 30 min. Samples were vortexed for 1 h with 11 μl of 10x loading dye followed by three cycles of 5 min incubations in dry ice/ethanol and at 65°C in a water bath. Samples were run on 0.7% agarose gel at 25-30 V overnight. DNA was transferred by capillary action to a positively charged nylon membrane (Roche), processed as per the manufacturer’s instructions, and exposed using a DIG-labelled probe (see DNA methods) and anti-DIG antibody (1:10000 (v/v), Roche, product 11093274910) before washing and visualisation.