Gel logic 200 imaging system

Ten microliters of each of the PCR products were mixed with 2 μL of the loading buffer and resolved on 2 % agarose gel in 1× TBE buffer at 125 V for 45 min. The gel was stained by the SYBR® safe DNA gel stain (Life Technologies), imaged (Gel logic 200 imaging system, Kodak, New York, USA) and interpreted.

The Fur-box binding activity of E. coli Fur was analyzed using the hinfI site protection assays (9 (link)). The Fur-box in the E. coli iucABCD promoter (5′-GAGAATCATTAGCATTCGC-3′) contains the restriction enzyme HinfI site (5′-GAATC-3′). Binding of Fur to the Fur-box protects the hinfI site (highlighted) from being cleaved by HinfI (9 (link)). The iucABCD promoter was synthesized (GenScript co.) and cloned into plasmid pUC19 via BamHI and HindIII sites to create pUC19-iuc. For the hinfI site protection assays, pUC19-iuc was preincubated with purified Fur proteins in 10 μl reaction solutions containing MgCl₂ (2 mM), NaCl (150 mM), bovine serum albumin (0.1 mg/ml), and Tris (20 mM, pH 8.0) for 10 min at room temperature. Restriction enzyme HinfI (0.5 unit) (New England Biolab co.) was then added to the reaction solutions. After incubation at 37 ^°C for 10 min, the reaction was stopped by adding 2 μl 6× loading buffer (New England Biolab co). The digested DNA products were separated on 1.5% agarose electrophoresis gel containing ethidium bromide (0.1 μg/ml) in 0.5× TAE (Tris-acetate-EDTA) buffer, run at 120 V for 35 min. The gel images were taken using the Kodak Gel Logic 200 Imaging System. The intensities of the DNA band on the agarose gels were quantified using ImageJ (NIH).

The DNA binding activity assay was carried out using a fluorescence labeled 40 mer (5′-F*-AATTGCGATCTAGCTCGCCAGUAGCGACCTT ATCTGATGA-3′). For single-stranded (ss) DNA binding assay, the 40 mer (0.5 μM) was incubated with increasing concentrations of protein in buffer containing Tris (20 mM, pH 8.0), NaCl (50 mM), β-mercaptoethanol (1 mM), MgCl₂ (1 mM), and bovine serum albumin (0.5 mg/mL). For double-strand (ds) DNA binding assay, the fluorescence labeled 40 mer was annealed to a complementary ssDNA in an annealing buffer containing Tris (50 mM, pH 8.0), NaCl (50 mM), and MgCl₂ (10 mM). Prepared dsDNA labeled with fluorescence was incubated with increasing concentrations of protein in buffer as described above. After incubation at room temperature for 15 min, samples were loaded on to a 0.6% agarose gel in TAE buffer. The agarose gel was run at 10 V per cm for 30 min at room temperature and photographed in a KODAK Gel Logic 200 Imaging System.

Torula yeast (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in RNase-free double-distilled deionized water at 1 mg/mL. To an equal volume of RNA was added a 1 mg/mL suspension also in RNase-free double-distilled water. The two were vortex mixed briefly for 10 to 15 s and exposed to physiological temperature and at 6, 12, 24, 36, 48 and 72 h, samples were removed and electrophoresed on 1% agarose/TAE, fluorescently stained with ethidium bromide and the band intensities determined on a Kodak Gel Logic 200 Imaging System (Rochester, NY, USA).

Genomic DNA was obtained from tail biopsies using Wizard^® SV Genomic DNA Purification System (Promega, Madison, WI, United States) according to the manufacturer’s protocol.
Simple PCR reactions were carried out using DNA Amplitools Master Mix (Biotools, Madrid, Spain), specific primers (400 nM each) and 5 μL of genomic DNA in a final volume of 25 μL. Animals were genotyped using specific primers for ^P2X7REGFP Fw 5′-CCTACGGCGTGCAGTGCTTCAGC-3′ and Rv 5′-CGGCGAGCTGCACGCTGCGTCCTC-3′; primers for J20 Fw 5′-GGTGAGTTTGTAAGTGATGCC-3′ and Rv 5′-TCTTCTTCTTCCACCTCAGC-3′. PCR was carried out over 40 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 45 s for EGFP primers or over 40 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 45 s for J20 primers.
PCR amplification products were electrophoresed on a 1.5% (w/v) agarose gel and stained with SYBR^® Safe DNA Gel Stain (Life Technologies, Carlsbad, CA, United States). PCR bands were visualized by gel imaging system Gel Logic 200 Imaging System (Kodak, Rochester, NY, United States).