Dneasy kit

DNA extraction of cell pellets containing O. tsutsugamushi was performed using four different methods: Alkaline lysis, the commercial Qiagen DNeasy kit, boiling in water or boiling in DMEM.
The protocol for DNA extraction by alkaline lysis (hotshot) was as follows. The cell pellet was resuspended in 20–80 μl alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA) and boiled at 95°C for 15–60 min. The sample was cooled to 4°C and neutralization buffer (40 mM Tris-HCl, pH 7–8) was added at an equal volume (20–80 μl). Smaller volumes were used where lower bacterial numbers were expected.
DNA extraction using the Qiagen DNeasy kit was performed following the manufacturer’s instructions. DNA extraction by boiling in water or DMEM was performed by resuspending cell pellets in 40 μl water or DMEM and boiling at 95°C for 30 min.
Extracted DNA samples were stored at-20°C as required. Where DNA extraction methods were being directly compared, identical cell pellets were resuspended in an equal final volume.

Genomic DNA was prepared from a 10 mL culture of MAP K10 in Middlebrook 7H9 supplemented with 10% Albumin Dextrose Catalase enrichment, 0.2% (vol/vol) glycerol, 0.05% (vol/vol) Tween 80 and 2 µg mL⁻¹ mycobactin j. Mycobacteria were harvested in early to mid log phase of growth and the cells were pelleted at room temperature for 5 min at 14,000 g. Pellets were re-suspended in ATL buffer (Qiagen DNeasy^® Blood & Tissue Kit) and the samples were transferred to Lysing Matrix B tubes (0.1 mm silica spheres). Samples were homogenized in a FastPrep™ FP120 cell disruptor at 3 × 20 s, Speed 6, followed by centrifugation at 14,000 g for 5 min. Supernatants were transferred to fresh sterile micro centrifuge tubes and Proteinase K (Qiagen DNeasy^® Kit) was added followed by incubation overnight at 56 ^oC. DNA was extracted using DNeasy^® Kit (Qiagen) according to the manufacturer’s protocol. Concentration (40 µg mL⁻¹) and purity of the DNA were determined using the NanoDrop and the absorbance ratios 260/280 and 260/230.

Mycobacteria were harvested in early to mid log phase of growth and the cells were pelleted at room temperature for 5 min at 14,000 g. Pellets were re-suspended in ATL buffer (Qiagen DNeasy^® Blood & Tissue Kit) and the samples were transferred to Lysing Matrix B tubes (0.1 mm silica spheres). Samples were homogenised in a FastPrep™ FP120 cell disruptor at 3 × 20s, Speed 6, followed by centrifugation at 14,000 g for 5 min. Supernatants were transferred to fresh sterile micro centrifuge tubes and Proteinase K (Qiagen DNeasy^® Kit) was added followed by incubation overnight at 56 °C. DNA was extracted using DNeasy^® Kit (Qiagen) according to the manufacturer’s protocol.

Bacterial DNA was extracted according to a published protocol (21 (link)). Previously frozen fecal samples (30 mg) were placed in 800 μL of lysis buffer (500 mM NaCl, 50 mM tris-HCl, 50 mM EDTA, and 4% SDS), homogenized for 5 min at speed level 12 using the Bullet Blender Storm Tissue Homogenizer (Next Advance), and incubated at 70°C for 20 min. Samples underwent centrifugation at 5,000 g for 5 min at room temperature. The supernatant was mixed with 200 μL of 10 mM ammonium acetate, incubated on ice for 5 min, and centrifuged at 16,000 g for 10 min at room temperature. An aliquot of supernatant (750 μL) was mixed with equal volumes of chilled isopropanol and incubated for 30 min on ice. Samples were centrifuged at 14,000 g and 4°C for 15 min to pellet DNA. Pellets were rinsed twice with 70% ethanol and re-suspended in 150 μL Tris-EDTA. Proteinase K (15 μL) and buffer AL (200 μL; DNeasy Kit, QIAGEN) were added and samples were incubated at 70°C for 10 min. Then 100% ethanol (200 μL) was added and samples were transferred to a QIAGEN DNeasy spin column and processed per the manufacturer’s instructions for DNA purification (DNeasy Kit, QIAGEN). Samples were eluted in 100 μL EB buffer (QIAGEN) and dsDNA yield was measured using Qubit dsDNA BR assay kits and fluorometry (Qubit 2.0, Life Technologies, Carlsbad, CA).