Absence RSL3 price of DNA was verified by qPCR. Each RNA sample (300 ng) was reverse transcribed using random hexamer oligonucleotides (Bioline, London, UK). Specific primers were designed to amplify an approximately 100 bp region of each gene in the study (Additional file 1: Table S1). qPCR was performed using a StepOnePlus™ Real-Time PCR System (Applied buy Barasertib Biosystems); each reaction consisted of 1 μl of cDNA, 1 x SensiMixPlus SYBR (Quantace, London, U.K.), 200 nM of specific primers
in a 25 μl reaction. The amplification cycling conditions were: initial denaturation at 95°C for 10 min; 39 cycles of denaturation at 95°C for 10 s; annealing at 60°C for 30 s; extension at 72°C for 5 s. A melting curve analysis was performed for each amplification reaction, with a temperature gradient of 0.1°C from 55°C to 95°C. No-template controls and a calibration curve, consisting of 6 dilutions of the selleck chemicals llc PCR amplicon of each gene cloned into PCR-Blunt vector (Invitrogen, Paisley,
U.K.) linearised with Nco I (NEB, Herts, U.K.), were included in every experiment (Additional file 1: Table S1). Statistical analysis was performed using a one-way ANOVA comparing gene copy numbers at different time points in each experiment to test the hypothesis that there is no variation in gene copy number during the recovery period. A post-hoc Dunnett’s test was employed, using the sample corresponding to the lysogen culture (-60) as the reference group, to assess whether or not time points
differed from the reference. P < 0.05 values were considered to be statistically significant. Protein extraction for 2D-PAGE Cultures of MC1061 and MC1061(Φ24B) were incubated for 6 h at 37°C. Cells were harvested and pellets washed in 1 ml of wash solution PIK3C2G (10 mM Tris-HCl, pH 8.0; 1.5 mM KH2PO4; 68 mM NaCl; 9 mM NaH2PO4). Cells were resuspended in 1 ml of resuspension buffer (10 mM Tris-HCl, pH 8.0; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM DTT; 0.1% SDS; 20 μl of protease inhibitor [Roche CompleteMini EDTA Free protease inhibitor cocktail tablets]) and each sample was sonicated for 5 × 10 s. DNase was added (5 μg ml-1) and samples were incubated for 1 h at 37°C. Samples were centrifuged for 1 h at 12,000 g, the supernatant recovered and protein concentration determined using the Bradford Assay. Aliquots (110 μg protein) of the sample were taken and precipitated in 10% TCA in acetone containing 20 mM DTT for 45 minutes at -20°C. Pellets were washed twice in ether. 2D-PAGE Isoelectric focussing was carried out on 18 cm IPG strips (pH 4-7,3-5.6 and 5.3-6.5;GE Healthcare), at 3,500 V for 7 h. Proteins were separated in the second dimension on 1.5 mm 4% stacking/15% resolving SDS-PAGE gels, for 6.5 h at 20 W per gel (up to maximum of 180 W). Proteins were silver stained .