Storm 860 phosporimager

DNA probes were generated by PCR using DNA primers based on the Mtb H37Rv sequences denoted in Tuberculist (34 (link)) with the addition of BamHI restriction sites for downstream cloning as needed. The PCR forward primer was labeled with [γ- ³³P]-ATP using T4 DNA polynucleotide kinase (New England Biolabs). DNA fragments were then labeled by PCR (30 cycles) using Mtb genomic DNA as a template, diluted 1:3 and 1 μl DNA probe was used in each 10 μl binding reaction. Samples were electrophoresed on an 8% (29:1) non-denaturing polyacrylamide gel for 2.5 h with a constant voltage of 150 v. Gels were vacuum dried, exposed on a phosphor screen, scanned with a Storm 860 PhosporImager (Molecular Dynamics), and analyzed with ImageQuant software (Molecular Dynamics).

PCR forward primers (Supplementary Table S3) were labeled with [γ- ³³P]-ATP (MP Biomedicals or Perkin Elmer) using T4 DNA polynucleotide kinase (New England Biolabs). DNA probes were generated using labeled forward primer and unlabeled reverse primer in PCR reaction. About 0.05 pmol DNA probe was used in each 10 μl binding reaction mixture. Briefly, 0.3 μM His-Cmr (N- or C-terminal tagged) and DNA probes were incubated at room temperature for 30 min in DNA binding buffer [10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1 mM EDTA, 50 μg/ml bovine serum albumin, 1 mM dithiothreitol, 0.05% non-ionic P-40 detergent, 20 μg/ml poly(dI-dC) and 10% glycerol], with or without 100 μM cAMP. Samples were loaded on an 6, 8 or 12% non-denaturing polyacrylamide gel, depending on the size of the DNA probe, and run for 2–3 h at 14 V/cm in 0.5× Tris-borate-EDTA buffer at 4°C. Gels were transferred to Whatman paper, vacuum-dried, exposed overnight on a phosphor screen, scanned with Storm 860 PhosporImager (Molecular Dynamics) and analyzed with ImageQuant software (Molecular Dynamics). About 200–500× excess competitor DNA was used wherever mentioned.