After generation of RACE-Ready cDNA, a PCR and a nested PCR were performed by using the inrR-specific primer 95,156rv plus the Universal Primer A (UPM, Clontech), HKI-272 and the

inrR primer 95,677rv plus the Nested Universal Primer A (NUP), respectively. Both PCR products were sequenced using a further inrR specific primer 95,790rv in the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems), and were separated on ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). A further successful mapping was deployed with 5′RACE on the transcript starting upstream of the most distal ICEclc ORF101284. 5′RACE reactions for the regions upstream of ORFs 58432, 66202, 73676, 81655, 88400, and 89746 did not produce specific fragments. Digoxigenin-labeled probe synthesis DNA regions of between 126 and 560 bp of 21 selected ORFs from the clc element’s core region (Figure 1) were amplified by PCR for probe synthesis (Additional file 1, Table S3). One of the PCR primers

(reverse complementary to the targeted ORF) included the sequence for the promoter region this website of T7 RNA polymerase. Antisense digoxigenin-labeled RNA probes were then synthesized from ~1 μg of purified PCR product by using T7 RNA polymerase according to instructions of the suppliers (Roche Applied Science). Northern hybridization 20 μg of total RNA were incubated in 20 μl (total volume) of denaturation buffer (containing 1 M glyoxal, 25% v/v learn more dimethylsulfoxide, 10 mM sodium phosphate, pH 7.0) for 1 h at 50°C. 100 ng of a digoxigenin-labeled RNA molecular weight marker I (0.3 — 6.9 kb, Roche Diagnostics)

was treated similarly. A volume of 0.2 μl of a 10 mg/ml ethidium bromide solution and 1 μl loading buffer (containing 50% sucrose, 15 mg/ml bromophenol blue in DEPC-treated H2O) were added to the samples at the end of the incubation period and mixed. Fragments were separated at 50 V on a 1% agarose gel in 10 mM sodium phosphate buffer (pH 7.0). RNA was subsequently transferred from gel Selleck Staurosporine onto Hybond N+ nylon membrane (Amersham Biosciences) in 10 × concentrated SSC solution (containing 3 M NaCl and 0.3 M sodium citrate dissolved in demineralized H2O) with the help of the VacuGene XL system (Amersham Biosciences) for 3.5 h at a vacuum of 50 mbar. After transfer, RNA was fixed to the membrane with a UV crosslinker (CX-2000, UVP) at a dose of 0.3 J per cm2. Immediately before hybridization, the membrane was rinsed with 20 mM Tris-HCl (pH 8.0) at 65°C for 10 min to remove glyoxal. The hybridization was performed in DIG Hybridization buffer (Roche Diagnostics) for 15 h at 68°C. The washing steps and the immuno-chemiluminescent detection were done according to the supplier’s instructions (Roche Diagnostics) using alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments and CSPD as reagent for the chemiluminescence reaction. Light emission was detected on Hyperfilm ECL (Amersham Biosciences).