Cdp star detection reagent

Genomic DNA was extracted from BFF cells using a NucleoSpin Tissue kit and PCR amplification was performed using PrimeStar HS DNA Polymerase (Takara Bio). For reverse transcription (RT)-PCR analysis, total RNA was prepared from cells using an RNeasy Micro kit (QIAGEN) with RNase-free DNase treatment. First-strand cDNA was synthesized from 1.0 µg of total RNA using a 1st strand synthesis kit (Takara Bio) and aliquots of 1/10-1/50 of the cDNA products were used for RT-PCR analysis. The PCR primers that were used for junction PCR and RT-PCR analysis are shown in Fig. 2a and Table S1. The PCR products were directly sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 310 Genetic Analyzer (Thermo Fisher Scientific). Homologous recombination was assessed by Southern blot analysis. Two µg of genomic DNA that had been digested with ScaI or BglII were separated on 0.8% agarose gels and transferred onto nylon membranes. Digoxigenin (DIG)-labelled DNA probes (Fig. 1a) were made by using PCR DIG Labelling Mix (Roche) with the primers listed in Table S1. Hybridization and DIG detection were performed using the DIG detection system (Roche) in accordance with the manufacturer’s protocol. The CDP-Star Detection Reagent (Roche) was used to develop the membrane and the resulting chemiluminescent signal was detected using Amersham Hyperfilm ECL (GE Healthcare).

Southern blotting was carried out according to the previously report34 (link) with some modifications, as described below. Five-μg aliquots of genomic DNA were digested with PstI, and 2 μg and 1 μg for the outer and the mNG probes, respectively, were resolved on 0.8% agarose gels. Digoxigenin-labelled DNA probes were made by PCR using KOD FX Neo (Toyobo) and DIG DNA labelling mix (Roche) with primers listed in Supplementary Table 6. Membrane transfer (Hybond-N+; GE Healthcare), ultraviolet cross-linking (120 mJ cm⁻²), pre-hybridization and hybridization were performed according to the instructions for DIG Easy Hyb Granules (Roche). The CDP-Star Detection Reagent (Roche) was used to develop the membrane, following the manufacturer’s instructions. The chemiluminescent signal was detected using Amersham Hyperfilm ECL (GE Healthcare).

EMSAs were performed using the DIG Gel Shift Kit (Roche) according to the manufacturer’s instructions. Probes used in this assay were designed from the possible transcription starting sites in the tiling microarray, and PCR-amplified from P. gingivalis ATCC 33277 DNA using primers shown in Supplemental Table 2. Labelled probes (4 ng) were incubated with rSigP (1 μg) in 20 μl binding buffer for 30 min at room temperature. The reactions were run on 6% Novex DNA gel Retardation gels in 0.5 × TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) at 4 °C. DNA–protein complexes were electrically transferred to Hybond-N⁺ membranes (GE Healthcare), and DIG-labelled reactions detected with chemiluminescence using an alkaline phosphatase-conjugated anti-DIG antibody (Roche) and CDP-star detection reagent (Roche).