7% agar and containing 100 μL of overnight bacterial culture was spotted after solidification with 5 μL suspensions of the 16 isolated bacteriophages. The plates were incubated at 25 °C, and the occurring lysis was investigated after 18–24 h. Propagation of phages for genome isolation was initiated according to the double agar layer method (Adams, 1959). After incubation at 25 °C for 18 h, the top layers were collected and placed into 10 mL SM buffer. ATR inhibitor After gentle agitation for 4 h at 25 °C, 2 mL chloroform was added, mixed, and incubated at 4 °C for 18 h. The resulting suspension was decanted over the chloroform and centrifuged at 5000 g for 40 min at 10 °C to eliminate the bacterial cells;
the supernatant was centrifuged again at 16 000 g for 60 min at 10 °C to collect the phage particles. The pellet was resuspended in 500 μL EDTA (pH 8.0) and 500 μL TES (10 mM Tris–HCl, 10 mM EDTA, 2% SDS, pH 8.0). After vigorous vortexing
for 30 s, the solution was incubated for 30 min at 65 °C. The proteins were precipitated with protein precipitation solution (Sigma-Aldrich), incubated on ice for 5 min, and centrifuged at 15 000 g for 4 min at 10 °C. The nucleic acid was precipitated with 0.1 volume 3 M Na-acetate and 1 volume ethanol. The pellet was washed with 70% ethanol; dried, and resuspended in 20 μL TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). To determine the type of the genome nucleic acid, RNase and DNase treatments were carried out according to the manufacturer’s (Sigma) instructions. Restriction fragment pattern differences of the investigated phage DNAs were examined BTK signaling pathway inhibitor with 21 different restriction endonucleases: AatI, AluI, ApaI, BamHI, BcuI, BglI, BglII, Bsh1236I, ClaI, DraI, EcoRI, HaeIII, Hin6I, HindIII, KpnI, MspI, NotI, PstI, RsaI, SacI, TaqI (Fermentas, Thermo Scientific). The growth Baf-A1 supplier characteristics of the Bf7 were investigated using the double layer method
(Adams, 1959) on its host, incubated for 18–48 h at different temperatures (5, 10, 20, 25, 30, and 35 °C), and the resulting plaque numbers and morphologies were compared. The single-step growth curve experiments were carried out according to the protocol of Keel et al. (2002), with minor modifications. The LB liquid medium was supplemented with glucose (0.3%), CaCl2 (0.075 mM), MgSO4 (2 mM), and FeCl2 (0.004 mM) according to the suggestions of Sambrook et al. (1989), for higher phage titer. Exponential-phase culture of P. tolaasii 2342T was treated with bacteriophage solution to have a multiplicity of infection (MOI) of 0.06. To visualize phage morphology with transmission electron microscopy, phage plaques were picked and placed in SM buffer. Aliquots were mounted on a carbon-coated formvar film supported by a 300 mesh copper grid. Samples were negatively stained with 1% uranyl acetate and examined by a Zeiss CEM 902 electron filtering electron microscope.