The reactions were performed as per the manufacturer’s instructions www.selleckchem.com/HIF.html (New England Biolabs, UK). DNA fragments were electrophoresed on 1% agarose gel at 5 V cm−1, in 1× TAE buffer (40 mM Tris, 40 mM acetate, 2 mM EDTA, pH 8.0). φ Lambda HindIII digest was used as DNA molecular weight marker. Gels were stained with ethidium bromide and photographed. Pulsed-field gel electrophoresis (PFGE) of bacteriophage DNA was carried out using the protocols described earlier (Carlson, 2005). Agarose plugs were prepared by mixing 1 mL of bacteriophage concentrate with 1 mL of 1.6% PFGE-grade agarose (Bio-Rad, Hercules, CA) and allowed to solidify at room temperature. These agarose

plugs were incubated in EDTA sarcosine proteinase K (ESP) (0.5 M EDTA, pH 9,

1% N-laurylsarcosine, and mg mL−1 proteinase K) overnight at 50 °C to digest the bacteriophage coat proteins and then washed thrice with TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5). The plugs were then treated with RNase A (μg mL−1) by incubating at 37 °C for 30 min. Restriction digestion was performed with various enzymes such as AluI, BamHI, BgII, DraI, HindIII, HaeII, KpnI, NcoI, NotI, PstI, XbaI, and ScaI following manufacturer’s instructions. The plugs were cut to 2-mm slices, placed in 1× restriction buffer, and incubated for 10 min in a 37 °C water bath. The buffer was removed, and fresh restriction buffer selleck containing 10 U of enzyme was added and incubated at 37 °C in water bath for 4 h. PFGE was carried out in 1% agarose gels in a BioRad CHEF DR-III PFGE

system (Bio-Rad), at 120° angle and 6 V cm−1, using ramped pulse times from 1 to 12 s for 6 h in 0.5× TBE (45 mM Tris, 45 mM borate, 1 mM EDTA pH 8.0) at 14 °C. Low-range PFGE marker was used as molecular weight size standard. Genome size was estimated by adding the length of each DNA fragment in the PFGE profile of ScaI and XbaI separately. The REA and PFGE patterns were captured using the Quantity one electrophoresis analysis system (Bio-Rad). Gel images were digitally normalized Protein kinase N1 to a single DNA marker to reduce gel-to-gel restriction pattern variability, and cluster analysis was carried out using molecular analyst software – Fingerprinting II (Version 3.0; Bio-Rad) by unweighted pair group method with arithmetic mean. Ability of the phages to transduce genetic elements was demonstrated by the transduction of the plasmid pHSG396 (Takara Bio Inc., Shiga, Japan), which possesses two selective phenotypic markers, β-galactosidase and chloramphenicol resistance. An isolate of V. harveyi (Vh57) susceptible to all four phages was transformed by CaCl2 treatment (Sambrook et al., 1989). Transformants carrying the plasmid were grown in 10 mL PYSS broth supplemented with 50 μg mL−1 chloramphenicol at 30 °C. This broth was suitably diluted with sterile PYSS to obtain 108 cells and mixed with four bacteriophage suspensions at a multiplicity of infection of one in separate tubes and incubated for 15 min at 30 °C.