1xtae buffer

The confirmation of phage classification to 936-type phages was performed on phage lysates (1 µl) as templates using multiplex PCR with 936- (5′-TCAATGGAAGACCAAGCGGA-3′/5′-GTAGGAGACCAACCCAAGCC-3′) and c2-specific (5′-CAGGTGTAAAAGTTCGAGAACT-3′/5′-CAGATAATGCACCTGAATCA-3′) primers, as described earlier^{56 (link)}. PCR products were separated on a 0.7% (w/v) agarose gel in 1xTAE buffer (Merck), stained with ethidium bromide, and visualized under UV light on a G:BOX instrument (Syngene).

Phage DNA digestion and agarose gel electrophoresis were carried out as previously established^{52 (link)}. Essentially, the genomic DNA of 90 phage isolates was cut separately with EcoRI, EcoRV or HindIII restriction endonucleases (Fermentas) overnight at 37 °C as recommended by the manufacturer. Enzymes were selected based on their ability to recognize and cleave within 6 nucleotide sites of low G-C content (33%), typical for lactococcal phages and their host genomes. The digested DNA fragments were separated on a 0.7% (w/v) agarose gel containing ethidium bromide in 1xTAE buffer (Merck). DNA loading dye with 50% formamide was used to load the samples on the agarose gel. Formamide was added to separate the phage cohesive (cos) ends^{53 (link)}. Comparative analysis based on DNA restriction patterns allowed the separation of the phages into distinct restriction groups (8 in total). Representatives from each group were taken for sequencing.