This process was repeated twice to ensure purity. Phage purity was confirmed using PCR assays. Amplification of phage stocks was achieved by modifying previous methods [53]. Briefly, mid-exponential phase PAO1 cultures (100 ml) were infected with purified LES phage (MOI = 0.1), at 37°C for 2 h. Lysed cultures were filter-sterilized. Electron microscopy Phage suspensions (1×109 – 1×1010 p.f.u. ml-1) were concentrated by centrifugation, negatively stained with 2% (w/v) uranyl acetate [54], and examined by transmission electron microscopy (magnification x 200,000). Multiplex PCR to confirm pure phage stocks and lysogens Three primer sets,

LESnest1 F/R, buy Entinostat Clust6nest F/R and 4tot1 F/R (Table 4), for the detection of LES phages 2, 3 and 4 respectively, were combined in a multiplex PCR selleck screening library assay for confirmation of each pure phage stock and each PLPL. Colony or filtered phage suspensions were used as templates in each reaction as described previously [25]. Table 4 Primer sequences Primer Sequence (5′-3′) Amplicon (bp) Cycling conditions Reference Multiplex PCR: LES1nestF tttggtgatgatcggcttagc 289 95°C,

4 min then 30 cycles: 95°C, 30 s; 58°C, 30 s; 72°C, 30 s; final extension step, 72°C, 7 min; [25] LES1nestR tgtggaagcgatcagtct       Clust6nestF ggatcgacgtggcataatctg 410   [25] Clust6nestR acgattctccggcatgcagcg       4tot1F gctcatgagtggctgacaac 105   This study 4tot1R tcttgggcagagaaccattc       Q-PCR: 2pro3F caagccctgtctggattttc 102 95°C, 10 min; then 40 cycles: 95°C, 10 s; 60°C, 15 s; 72°C s. This study 2pro3R gagacaggttgggagggagt       3tot1F cgcaggtaccaccagacttt 122   This study 3tot1R catgtccagcaggttcaaaa       3pro3F gcggatgttctcaaacgaat GF120918 ic50 134   This study 3pro3R cgggagaagcaatgacctac     Casein kinase 1   4tot1F gctcatgagtggctgacaac 105   This study 4tot1R tcttgggcagagaaccattc       4pro3F tcgtgctgtgctgatctttt 172   This study 4pro3R agcagtgccagttgatgttg       Preparation of DIG-labeled probes: φ2intDIGF tgcctatctaacggggttca 1097 95°C, 4 min. 30 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 1 min s; final extension step, 72°C, 7 min This study φ2intDIGR gaagcaaccgagaagtggag     φ3intDIGF ggatcatgtagcgggaaaga 874 This study φ3intDIGR agaacctggcgaaagtctga     φ4cIDIGF atcgttaattggcacggaat

893 This study φ4cIDIGR acagcaacggatttccactc     tot = to quantify total phage copies; pro = to quantify total phage copies. Quantifying production of each LES phage from LESB58 Replication of each LES phage in response to induction of the lytic cycle was compared using Q-PCR to distinguish and enumerate each specific phage type. LESB58 induction experiments were performed on three separate occasions in the presence and absence of norfloxacin for 30 and 60 min exposure times before the 2 h recovery step. DNA was prepared from each replicate using the Bacterial and Virus DNA extraction kit (QIAGEN) and the automated QIAsymphony machine (QIAGEN; pathogen complex 200 protocol). Q-PCR was performed using six specific primer sets to differentiate between prophage and total copies of each phage.