Additional file 11 of Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis

Additional file 11: Figure S6. Purification of host cell-free Ehrlichia sp. HF and bacterial genomic DNA. (A) Twelve T175 flasks of Ehrlichia sp. HF-infected DH82 cells (>80% infectivity) at 3d pi were homogenized in 30 ml of 1× SPK for 30 times with type B tight-fitting pestle. Pellet following centrifugation at 700 × g was homogenized for additional 30 times. Both homogenates were step-wise centrifuged at 700, 1,000, and 1,500 × g, passed through 5.0- and 2.7-μm filters, and centrifuged at 10,000 × g for 10 min. Host cell-free Ehrlichia sp. HF was purified with very low host nuclear contamination under Diff-Quik staining. Bar, 10 μm. (B) Genome DNAs of Ehrlichia sp. HF (EHF) were purified using Qiagen genomic tips and dissolved in TE buffer. DNAs were resolved using 0.9% agarose with BioLine molecular weight (MW) markers with DNA concentrations of each band showing inside parenthesis. Genomic DNA bands above 20 kB were visible, and the concentration was above 15 ng/μl. PCR reactions were carried out with 35 cycles at 98°C for 30-second, 60°C for 30-second, and 68°C for 1-minute. Primers targeting 16S rRNA gene of Ehrlichia sp. HF detected specific bands at 1/100 dilutions, but dog G3PDH primers did not amplify any bands under any dilutions (Dilutions: .1, 1/10, .01, 1/100 dilution; Pos.: positive control using DNA isolated from dog DH82 cells; -, negative control without DNA input).