0.22 μm pore sized filters

For isolation and analysis of phage nucleic acids, culture supernatants containing phage particles were filtered through 0.22 μm pore-sized filters (Millipore). The filtrates were mixed with one-fourth volume of a solution containing 20% polyethylene glycol (PEG-6000) and 10% NaCl, and centrifuged at 12000 x g to precipitate phage particles. The precipitate was dissolved in a solution containing 20 mM Tris-Cl (pH 7.5), 60mM Kcl, 10mM MgCl, 10mM NaCl, and digested with pancreatic DNAseI (100 units/ml) and RNAse A (50 μg/ml) at 37^°C for 2 hours. The solution was extracted with phenol-chloroform, and the total nucleic acids were precipitated with ethanol. Phage nucleic acids were suspended in deionized water and purified using the SV Minipreps DNA purification system (Promega Madison, USA). The phage nucleic acid was digested with restriction endonucleases (Invitrogen Corporation, Carlsbad, CA) and analyzed by agarose gel electrophoresis following standard procedures to initially check for diversity and select different phages for sequencing.

The samples positive for phages were then serially diluted, mixed with soft agar, and overlaid on hard bottom agar and incubated for 16–17 h at 37°C as described above. Next day, lytic plaque zones were collected by cutting the soft layer from the plate using sterile cut tips and placing them separately in 500 μl of logarithmic-phase cells, vortexed, and incubated at 37°C for 20 min. Then 3 ml of LB broth was added and incubated overnight at 37°C. Then, the culture was centrifuged at 10,000 × g for 20 min, and the supernatant was filtered through 0.22-μm pore-sized filters (Millipore) to exclude bacteria. The number of phage particles in the filtered supernatant was determined by testing serial dilutions of the supernatant with the host strain. This method was repeated for three successive times to obtain purified phages.

For isolation and analysis of phage nucleic acids, culture supernatants containing phage particles were filtered through 0.22 μm pore-sized filters (Millipore). The filtrates were mixed with one-fourth volume of a solution containing 20% polyethylene glycol (PEG-6000) and 10% NaCl, and centrifuged at 12000 x g to precipitate phage particles. The precipitate was dissolved in a solution containing 20 mM Tris-Cl (pH 7.5), 60 mM Kcl, 10 mM MgCl, 10 mM NaCl, and digested with pancreatic DNAseI (100 units/ml) and RNAse A (50 μg/ml) at 37 °C for 2 hours. The solution was extracted with phenol-chloroform, and the total nucleic acids were precipitated with ethanol. Phage nucleic acids were suspended in deionized water and purified using the SV Minipreps DNA purification system (Promega Madison, USA). The phage nucleic acid was digested with restriction endonucleases (Invitrogen Corporation, Carlsbad, CA) and analyzed by agarose gel electrophoresis following standard procedures to initially check for diversity and select different phages for sequencing.