Dye primer based sequencing kit

In order to determine the binding sites of SbbR on P_milR and P_R-A, DNase I footprinting assays were carried out as described previously (Li et al., 2016 (link)). DNA probes were prepared by PCR using 5′ FAM fluorescence-labeled primers listed in Supplementary Table S1. After being purified from agarose gel, labeled DNA probe (200 ng) and protein with different concentrations of SbbR-His₆ were added to a final reaction volume of 50 μl, and incubated for 25 min at 25°C. DNase I (Promega) digestions were carried out for 75 s at 25°C and stopped with 20 mM EGTA. After ethanol extraction and precipitation, the purified samples were loaded in an Applied Biosystems 3730 DNA genetic analyzer together with the internal-lane size standard ROX-500 (Applied Biosystems). The dye primer-based sequencing kit (Thermo) was used to further precisely determine the sequences after aligning the capillary electrophoresis results of reactions. The results were then processed with GeneMarker v2.2.0 software.

DNase I footprinting assays were performed based on a fluorescence labeling procedure56 (link). Briefly, the promoters DNA of exosporium genes were PCR-amplified using the fluorescently labeled primers and purified from an agarose gel. The labeled DNA probe (400 ng) was incubated for 30 min at 25 °C with the different amounts of GerE in a total volume of 40 μl binding buffer (described above for EMSA). DNase I digestion was then performed for 1 min at 25 °C and stopped with stop buffer (Promega). After phenol-chloroform extraction and ethanol precipitation, the samples were loaded on an Applied Biosystems 3730 DNA genetic analyzer with an internal-lane size standard (ROX-500, Applied Biosystems). A dye primer-based sequencing kit (Thermo) was used to precisely determine the sequences after their alignment wtih capillary electrophoresis results. Electropherograms were analyzed with GeneMarker v1.8 (Applied Biosystems).

DNase I footprinting assays were performed based on a fluorescent labeling procedure [23 (link)]. Briefly, gabT promoter DNA was PCR-amplified using the fluorescently-labeled primers PgabT-F and PgabT-R (Table 2) and purified from an agarose gel. The labeled PgabT DNA probe (120 ng) was incubated for 20 min at 25°C with the indicated concentrations of purified GabR and BSA in a total volume of 50 μl of binding buffer (described above for EMSA). DNase I digestion was then carried out for 1 min at 25°C and stopped with stop buffer (Promega). After phenol-chloroform extraction and ethanol precipitation, the samples were loaded on an Applied Biosystems 3730 DNA genetic analyzer together with an internal-lane size standard (ROX-500, Applied Biosystems). A dye primer-based sequencing kit (Thermo) was used to precisely determine the sequences after their alignment wtih the capillary electrophoresis results from the reactions. Electropherograms were analyzed with GeneMarker v1.8 (Applied Biosystems).