Rotihybrid quick buffer

Phage plaques were transferred to charged nylon membranes (GE Healthcare, Little Chalfont, UK) and screened with ³²P-labeled nucleic acid probes prepared using the DecaLabel kit (Thermo Fisher Scientific, Waltham, MA, USA). For pre-hybridization and hybridization, the membranes were incubated in RotiHybrid Quick buffer (Carl Roth, Karlsruhe, Germany) supplemented with 0.1 mg/mL salmon sperm DNA. The membranes were hybridized for 15 h at 65 °C and washed four times with RotiHybrid Quick buffer diluted in progressively larger volumes of water (1:2 dilution for 30 min, 1:5 dilution for 30 min, and 1:10 dilution for 2 × 10 min). The membranes were exposed to X-ray film at − 80 °C for 4 days. Isolated positive cosmids were sequenced by primer walking, and the inserts were amplified and subcloned for DNA sequencing using the Zero Blunt Cloning Kit (Invitrogen, Karlsruhe, Germany).

Total RNA was isolated from cultures using the TRIzol Reagent (Ambion) according to the manufacturer's instructions. For Northern-blot analysis, 10 μg of total RNA were heated at 65°C for 5 min in loading buffer (5 mM EDTA, 0.025% xylene cyanol, 0.025% bromophenol blue dissolved in formamide) and resolved on 8% polyacrylamide/8 M urea gels. The RNA was transferred onto Hybond N+ nylon membranes (GEHealthcare) using a semi-dry electroblotting apparatus (Transblot SD cell, BioRad) set at 13 V for 45 min followed by UV-cross-linking. A DNA oligonucleotide probe specific for PaiI (Supplementary Table S2) was 5′-end labeled with [γ-³²P] ATP using T4 polynucleotide kinase. The [³²P]-labeled oligonucleotide was heated at 95°C for 2 min, added to pre-hybridized membranes and incubated at 52°C overnight. Pre-hybridization and hybridization were performed in Roti® Hybrid Quick buffer (Carl Roth, Karlsruhe, Germany) supplemented with 0.1 μg/ml salmon sperm DNA. A 5S rRNA-specific oligonucleotide was used as a loading control, (Supplementary Table S2). The signals were visualized using a PhosphorImager (Molecular Dynamics).