Random primer kit

Total RNA was isolated from 30 mL samples of Synechocystis cultures in the mid-exponential growth phase (3 to 4 µg chlorophyll mL⁻¹). Extractions were performed by vortexing cells in presence of phenol-chloroform and acid-washed baked glass beads (0.25–0.3 mm diameter) as previously described [47] (link). 5 µg of total RNA was loaded per lane and electrophoresed in 1.2% agarose denaturing formaldehyde gels [90] and transferred to nylon membranes (Hybond N-Plus; GE Healthcare). Prehybridization, hybridization, and washes were in accordance with GE Healthcare instruction manuals. Probes for Northern blot hybridization were synthesized by PCR using oligonucleotide pairs: petEF-petER, petJF-petJR, copM1F-copM1R, copBF-copBR, slr2015F-slr2015R, slr1667F-slr1667R, nrsBF-nrsBR, NRP3-NRP1, sufRF-sufRR, sufBF-sufBR (see Table S7) for petE, petJ, copM, copB, slr2015, slr1667, nrsB, 5′nrsD, sufR and sufB, respectively. As a control, in all cases the filters were stripped and re-probed with a 580-bp HindIII-BamHI probe from plasmid pAV1100 containing the constitutively expressed RNase P RNA gene (rnpB) from Synechocystis (Vioque, 1992). DNA probes were ³²P labeled with a random-primer kit (Amersham Biosciences) using [α-³²P] dCTP (3,000 Ci/mmol). Hybridization signals were quantified with a Cyclone Phosphor System (Packard). Each experiment was performed at least two independent times.