A fresh voided stool specimen was processed for total DNA isolation. Approximately 100 mg of stool was transferred to an Eppendorf safe lock tube and processed with a PowerSoil DNA isolation kit (Catalogue # 12888-100, MO BIO Laboratories, Inc) using the manufacture’s protocol with slight modifications. For efficient lysis of the microbes, glass beads in 200 µL bead solution plus 200 µL of Phenol/Chloroform/Isoamyl alcohol (25:24:1) pH 7.8–8.2 (Catalogue #327115000, Acros Organics) was added to the sample. The contents were vortexed for 1–2 min and homogenized at speed 10 for 5 min with air-cooling using the Bullet Blender Storm Homogenizer (Catalogue # BBY24M, Next Advance Inc). The contents were centrifuged at 14,000×g, and the lysate was transferred to a sterile tube. The DNA was eluted with 1×TE, pH 8.0, and stored at −80°C. The quality and quantity of the DNA was accessed using a spectrophotometer (NanoPhotometer Pearl, Denville Scientific, Inc), agarose gel electrophoresis, and fluorometer (Qubit® dsDNA High Sensitivity and dsDNA Broad Range assay, Life Technologies Corporation).
Qubit dsdna high sensitivity and dsdna broad range assay
Manufactured by Thermo Fisher Scientific
The Qubit® dsDNA High Sensitivity and dsDNA Broad Range assays are fluorescence-based assays designed for the accurate quantitation of double-stranded DNA (dsDNA) samples. The High Sensitivity assay is suitable for samples with concentrations between 0.2-100 ng/mL, while the Broad Range assay is suitable for samples with concentrations between 2-1000 ng/mL. Both assays provide a sensitive and specific method for determining dsDNA concentrations.
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2 protocols using qubit dsdna high sensitivity and dsdna broad range assay
1
Optimized stool DNA isolation protocol
2
Fecal and Cecal Microbiome Collection
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Mice were placed on wire bottom cages for 24 h to collect fecal pellets and stored at −80 °C. Following this, mice were anesthetized and dissected. Fresh cecal content was collected from the gut and placed into autoclaved microcentrifuge tubes, and stored at −80 °C till further use. One or two fecal pellets, and 25–50 mg of cecal content was used for metagenomic DNA isolation using PowerSoil DNA isolation kit (Catalogue # 12888-100, MO BIO Laboratories, Inc) using the method as described previously55 (link). The quality and quantity of the DNA was accessed using a spectrophotometer (NanoPhotometer Pearl, Denville Scientific, Inc), agarose gel electrophoresis, and fluorometer (Qubit® dsDNA High Sensitivity and dsDNA Broad Range assay, Life Technologies Corporation).
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