We recommend you start with 50–200 mg of stool. Homogenize each sample in a 2 ml bead beating tube containing a mixture of lysis beads. The combination of mechanical bead beating and proven lysis chemistry facilitates optimized lysis of host and microbial cells. Remove common inhibitors found in stool samples, and which interfere with PCR and other downstream applications with second generation IRT. Pass the lysate through an MB Spin Column in combination with a high-salt buffer, to allow selective, efficient genomic DNA binding. Wash and elute the DNA. It is ready for PCR analysis and other downstream applications, including qPCR and NGS.
Add Ethanol to the flow-through from the MB Spin Column to create appropriate RNA-binding conditions. Transfer the sample to an MB RNA Spin Column, for total RNA binding to the membrane. Wash and elute the RNA in RNase-free water. It is now ready for downstream applications including RT-PCR, qPCR and NGS.
Sample storage, stabilisation and preservation
The time between sample collection and preservation is among the main conditions to influence the yield and integrity of nucleic acids isolated from microbes in stool samples, together with the state of the digestive system and diet of the individua. We recommend that you process the sample as quickly as possible after collection, to optimize the quality of nucleic acids from stool. The PowerProtect DNA/RNA reagent enables stabilizes of stool samples at room temperature.