Bead disruption, inhibitor-oriented lysis and silica column purification for stool DNA extraction
Cat. No. D314101F / D314102F / D314103F
Representative workflow for ≤200 mg stool samples, liquid stool samples or stool suspensions in preservation solution.
Transfer 50–200 mg stool sample, 100–200 μl liquid sample, or 200–300 μl stool suspension containing preservation solution into the 2 ml Bead Tube.
Avoid transferring undigested food particles, seed shells or coarse debris. For dry, animal-derived or inhibitor-rich samples, a lower starting amount is usually safer.
Add 600 μl Buffer STL, 60 μl Buffer SL and 300 μl Buffer PCI. Screw the cap tightly before disruption.
This stage prepares the stool matrix for mechanical disruption and helps manage inhibitors commonly present in fecal material.
Process the bead tube using a rapid bead grinder. PowerLyzer and FastPrep-type routes are typically based on short grinding cycles with a pause and repeat.
The displayed cumulative timeline uses the rapid bead-grinding route. Vortex-based disruption takes 10 minutes, and Tissue Lysis II handling may be longer depending on repositioning; this variation is reflected in the final total-time range.
Centrifuge at 14,000 × g for 10 minutes at room temperature to pellet stool debris and bead-associated material.
A clean clarified supernatant is important for column flow. Excess solids or viscous lysate increase the risk of clogging in the downstream silica membrane step.
Transfer 600 μl supernatant into a new 2 ml centrifuge tube. Add 600 μl Buffer GWP and 20 μl Proteinase K, invert 6–8 times, and stay at room temperature for 10 minutes.
If DNA yield is expected to exceed the column capacity under GWP mediation, isopropanol may be used as described in the manual. Do not add isopropanol when the mixture is darkly colored because pigment or impurity precipitation may increase.
Install a HiPure DNA Mini Column II in a 2 ml collection tube. Transfer half of the mixture to the column and centrifuge at 13,000 × g for 1 minute.
The timeline includes handling time for loading, centrifugation and column repositioning.
Discard filtrate, reinstall the column and transfer the remaining mixture. Centrifuge at 13,000 × g for 1 minute.
Repeated loading is part of the standard column route because the binding mixture exceeds a single loading volume.
Add 600 μl Buffer GWP to the column and centrifuge at 13,000 × g for 1 minute.
This wash supports removal of stool-derived matrix components before ethanol-containing wash steps.
Add 600 μl Buffer GW2 and centrifuge at 13,000 × g for 1 minute.
Buffer GW2 must be prepared with absolute ethanol before use. Incorrect ethanol addition is a common cause of poor wash performance.
Repeat the GW2 wash once to further reduce residual salts and inhibitory components.
For stool DNA, wash completeness and filtrate removal matter more than adding extra unplanned steps.
Discard filtrate, reinstall the column and centrifuge at 13,000 × g for 2 minutes to dry the membrane.
Drying helps remove residual ethanol. Incomplete drying may affect downstream reactions such as PCR or ligation-sensitive applications.
Place the column in a 1.5 ml centrifuge tube. Add 50–100 μl Elution Buffer preheated to 65°C to the center of the membrane, stand for 3 minutes and centrifuge at 13,000 × g for 1 minute.
The elution volume can be adjusted according to downstream concentration needs, provided the final eluate volume remains compatible with the assay.
Transfer the eluate back to the center of the column membrane, stand for 3 minutes and centrifuge at 13,000 × g for 1 minute.
Re-elution improves recovery from the same membrane without changing the downstream column chemistry.
Discard the column. Store purified DNA at 2–8°C for short-term use or at -20°C for long-term storage.
Purified DNA is suitable for downstream molecular applications such as PCR, Southern hybridization, enzyme digestion and next-generation sequencing.
This workflow separates stool-specific sample pretreatment from the shared silica column purification path. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. The workflow shown here focuses on the standard stool matrix route using bead-tube disruption, inhibitor-oriented lysis chemistry, clarified supernatant transfer and downstream spin-column purification.
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifuge handling, filtrate removal and tube or column repositioning. For short protocol ranges, the timeline uses the midpoint. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. Cumulative time runs continuously from sample loading to final elution across all workflow sections and is displayed in each step as the running total for the standard route. Downstream column steps include handling time for repeated loading, buffer addition, filtrate disposal and column repositioning.
D3141F is not a simple direct lysis workflow. Stool contains particulate material, mucus, polysaccharides, bile pigments, humic-like inhibitors and variable water content. The workflow first uses bead-tube disruption and inhibitor-control chemistry to release DNA and reduce matrix interference, then transfers clarified supernatant into a GWP / Proteinase K binding-preparation step before silica membrane purification.
The key handling point is controlling the stool matrix before the column stage. Do not transfer coarse undigested particles into the bead tube. For animal stool, very dry stool or samples rich in lipid, polysaccharide or protein, reducing the starting amount may improve lysis efficiency, membrane flow and DNA purity. For liquid stool or stool preserved in solution, the combined residue and residual liquid should not exceed the recommended working volume. Darkly colored mixtures should not be treated with isopropanol unless the manual conditions are clearly appropriate, because pigment and impurity precipitation can increase membrane burden.