T7 sequenase 2.0 dna sequencing kit

DNase I foot printing assay was conducted as described (41 (link)). DNA fragments corresponding to the lpxT region were generated by PCR using primer W3436 and ³²P-labeled primer W3435 with genomic DNA from strain 14028s as template. Unincorporated [γ-³²P]-ATP was removed by using G-50 microcolumns (GE Healthcare). A total of 2 × 10⁴ cpm of labeled probe (~10 fmol), 200 ng of poly (dI-dC) (Sigma), and purified His-tagged PhoP (PhoP-6xHis) (49 (link)) were mixed with binding buffer [20mM Hepes at pH8.0, 10mM KCl, 2mM MgCl₂, 0.1 mM EDTA, 0.1 mM DTT, 50 µg·mL⁻¹ BSA, and 10% (vol/vol) glycerol] in a total volume of 20 µL and incubated at room temperature for 20 min. DNase I (GIBCO) (0.01 units), 10 mM CaCl₂, and 10 mM MgCl₂ were added and incubated at room temperature for 3 min. Final concentrations of PhoP protein were 0, 1, 5, 10, 20 µM. The reaction was stopped by the addition of 100 µL of a phenol chloroform solution, and the aqueous phase was precipitated with ethanol. The precipitate was dissolved in sequence-loading buffer and electrophoresed on a 6% acrylamide/7 M urea gel together with a sequence ladder generated with the labeled primer by using the T7 Sequenase 2.0 DNA-sequencing kit (Amersham Biosciences), and the gels were dried and autoradiographed.